Ovarian cancer vaccines

ABSTRACT

Provided herein are methods, kits and compositions for the treatment and/or prevention of ovarian cancer through the induction of an immune response against Anti-Mullerian Hormone Receptor, Type II (AMHR2).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofPCT/US2016/050159, filed Sep. 2, 2016, which claims the benefit ofpriority to U.S. Provisional Patent Application No. 62/213,286, filedSep. 2, 2015, which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 18, 2018, isnamed SBS-00301_SL.txt and is 12,214 bytes in size.

BACKGROUND

Epithelial ovarian cancer (EOC) is the leading cause of death fromgynecologic malignancies in the United States. Approximately 60% ofovarian cancers are diagnosed at late stages, and although initialresponses to the current standard of care are high, most patients havedisease recurrence resulting in a five year overall survival (OS) rateslightly over 45%.

Induction of ovarian tumor immunity through vaccination is a promisingapproach, the potential efficacy of which is supported by the increasedOS observed in patients whose ovarian tumors are infiltrated by T cells.Therapeutic ovarian cancer vaccine strategies using whole tumorhomogenate or certain tumor associated antigens, such as human epidermalgrowth factor receptor 2 (HER2), cancer-testis antigen 1 (CTAG1B orNY-ESO-1), and cancer antigen 25 (CA-125) have been attempted, but haveprovided only modest therapeutic results (Zhang et al., Journal ofMedicine 348:203-213 (2003); Kandalaft et al., Journal of ClinicalOncology 29:925-933 (2011); Bookman et al., Journal of Clinical Oncology21:283-290 (2003); Odunsi et al., Cancer Research 63:6076-6083 (2004);and Rosen et al., Gynecologic Oncology 99:267-277 (2005), each of whichis hereby incorporated by reference in its entirety).

Thus, considering the high rate of ovarian cancer recurrence and the lowfive year survival rate, there is great need for new compositions andmethods for the treatment and prevention of ovarian cancer.

SUMMARY

In certain aspects, provided herein are methods and compositions for thetreatment and/or prevention of ovarian cancer through the induction ofan immune response against Anti-Mullerian Hormone Receptor, Type II(AMHR2).

In certain aspects, provided herein is a method of treating an ovariancancer tumor (e.g., a primary or metastatic ovarian cancer, such as anepithelial ovarian cancer tumor) in a subject (e.g., a female humansubject) comprising administering to the subject an immunogeniccomposition comprising a polypeptide (i.e., an “AMHR2 polypeptide”)having an amino acid sequence that includes at least a portion of theamino acid sequence of an AMHR2 protein (e.g., at least a portion of SEQID NO: 1), a nucleic acid encoding an AMHR2 polypeptide, anantigen-presenting cell presenting an AMHR2 epitope, and/or anAMHR2-primed lymphocyte (e.g., a T lymphocyte and/or a B lymphocyte). Insome embodiments, the ovarian cancer tumor expresses AMHR2. In someembodiments, administration of the immunogenic composition induces animmune response against the ovarian cancer tumor in the subject (e.g., aT cell immune response, such as a type 1 and/or type 17 immune responseand/or a B cell immune response, such as an IgG response). In someembodiments, the subject is administered multiple doses of theimmunogenic composition (e.g., at least 2, 3, 4, 5 or 6 doses). In someembodiments, the subject has undergone surgery to remove at least partof the ovarian cancer tumor. In some embodiments, the method furthercomprises the step of surgically removing at least part of the ovariancancer tumor.

In certain aspects, provided herein is a method of preventing ovariancancer and/or preventing recurrence of ovarian cancer in a subject(e.g., a female human subject) comprising administering to the subjectan immunogenic composition comprising a polypeptide (i.e., an “AMHR2polypeptide”) having an amino acid sequence that includes at least aportion of the amino acid sequence of an AMHR2 protein (e.g., SEQ ID NO:1), a nucleic acid encoding an AMHR2 polypeptide, an antigen-presentingcell presenting an AMHR2 epitope, and/or an AMHR2-primed lymphocyte(e.g., a T lymphocyte and/or a B lymphocyte). In some embodiments,administration of the immunogenic composition induces an immune responseagainst the AMHR2 in the subject (e.g., a T cell immune response, suchas a type 1 and/or type 17 immune response and/or a B cell immuneresponse, such as an IgG response). In some embodiments, the subject isadministered multiple doses of the immunogenic composition (e.g., atleast 2, 3, 4, 5 or 6 doses). In some embodiments, the subject hasundergone surgery to remove at least part of an ovarian cancer tumore.g., an ovarian cancer tumor expresses AMHR2, such as a primary ormetastatic ovarian cancer tumor). In some embodiments, the tumor is anepithelial ovarian cancer tumor. In some embodiments, the method furthercomprises the step of surgically removing at least part of the ovariancancer tumor.

In some embodiments of the methods provided herein, the immunogeniccomposition comprises an adjuvant. In some embodiments, the adjuvant isAdjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calciumphosphate, β-Glucan Peptide, CpG DNA, GM-CSF, GPI-0100, IFA, IFN-γ,IL-17, lipid A, lipopolysaccharide, Lipovant, Montanide,N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, poly-IC, quil A,trehalose dimycolate, or zymosan. In some embodiments, the adjuvant isone that induces a mixed type 1/type 17 immune response.

In some embodiments of the methods provided herein, the method furthercomprises the step of determining whether an ovarian cancer tumor in thesubject and/or from the subject expresses AMHR2. In some embodiments,the methods include the step of testing a tumor sample obtained from thesubject for expression of AMHR2. In some embodiments, expression ofAMHR2 is determined by detecting the presence of AMHR2 protein in thetumor sample (e.g. by FACS, fluorescent microscopy, western blot, etc.).In some embodiments, the AMHR2 protein is detected by contacting thesample with a detectably labeled antibody (e.g., an antibody directly orindirectly labeled with a fluorescent moiety). In some embodiments,expression of AMHR2 is determined by detecting the presence of AMHR2mRNA (e.g., by RT-PCR, northern blot, etc.). In some embodiments, theAMHR2 mRNA is detected by contacting the sample with a detectablylabeled nucleic acid probe (e.g., a probe directly or indirectly labeledwith a fluorescent moiety). In some embodiments, the method comprisedobtaining the sample from the subject (e.g., by tumor biopsy).

In some embodiments of the methods provided herein, the method furthercomprises the step of determining whether the administration of theimmunogenic composition induces an immune response against the ovariancancer tumor and/or AMHR2 in the subject. In some embodiments, theimmune response is a T cell immune response (e.g., a mixed type 1/type17 immune response). In some embodiments, the immune response isdetected by detecting the presence of cytokines (e.g., IFN-γ and/orIL-17) in a patient sample (e.g., a patient serum sample). In someembodiments, the cytokines are detected by contacting the sample with,directly or indirectly, detectably labeled antibodies specific for thecytokines to be detected. In some embodiments, the cytokines aredetected by performing an ELISA assay. In some embodiments, the immuneresponse is a B cell immune response. In some embodiments, the B cellimmune response is detected by detecting the presence of anti-AMHR2antibodies e.g., anti-AMHR2 IgG antibodies) in a patient sample (e.g., aserum sample).

In certain embodiments, the methods provided herein further comprise thestep of administering an additional agent to the subject. In someembodiments, the additional agent is an anti-cancer agent. In someembodiments, the anti-cancer agent is paclitaxel, cisplatin, topotecan,gemcitabine, bleomycin, etoposide, carboplatin, docetaxel, doxorubicin,topotecan, cyclophosphamide, trabectedin, olaparib, tamoxifen, letrozoleor bevacizumab. In some embodiments, the anti-cancer agent is an immunecheckpoint inhibitor. In some embodiments, the immune checkpointinhibitor is an inhibitor of CTLA4, such as an anti-CTLA4 antibody(e.g., ipilimumab (BMS), tremelimumab (AstraZeneca) and/or KAHR-102(Kahr Medical)). In some embodiments, the immune checkpoint inhibitor isan inhibitor of PD-1, such as an anti-PD-1 antibody (e.g., nivolumab(BMS), pembrolizumab/lambrolizumab (Merck), pidilizumab (Curetech),AMP-224 (GSK), AMP-514 (AstraZeneca), STI-A1110 (Sorrento) and/orTSR-042 (Tesaro). In some embodiments, the immune checkpoint inhibitoris an inhibitor of PD-L1 and/or PD-L2, such as an anti-PD-L1 and/or ananti-PD-L2 antibody (e.g., RG-7446 (Roche), BMS-936559 (BMS), MEDI-4736(AstraZeneca), MSB-0020718C (Merck), AUR-012 (Pierre Fabre Med),STI-A1010 (Sorrento)).

In certain embodiments of the methods provided herein, the human femalesubject is predisposed to ovarian cancer. In some embodiments, thegenome of the subject comprises a BRCA1 or BRCA2 mutation thatpredisposes the subject to ovarian cancer. In some embodiments, thesubject has a family history of ovarian cancer. In some embodiments, thesubject is a post-menopausal human female.

In certain aspects, provided herein is a kit and/or a composition (e.g.,an immunogenic composition) for preventing and/or treating ovariancancer in a subject (e.g., a female human subject), the kit orcomposition comprising a polypeptide (i.e., an “AMHR2 polypeptide”)having an amino acid sequence that includes at least a portion of theamino acid sequence of an AMHR2 protein (e.g., SEQ ID NO: 1), a nucleicacid encoding an AMHR2 polypeptide, an antigen-presenting cellpresenting an AMHR2 epitope, and/or an AMHR2-primed lymphocyte (e.g., aT lymphocyte and/or a B lymphocyte). In some embodiments, the kitcomprises multiple doses of the polypeptide, nucleic acid, antigenpresenting cell and/or lymphocyte. In some embodiments, the kit furthercomprises instructions for use.

In certain embodiments of the kits and compositions provided herein, thekit or composition further comprises an adjuvant. In some embodiments,the adjuvant is Adjuvant 65, α-GalCer, aluminum phosphate, aluminumhydroxide, calcium phosphate, 13-Glucan Peptide, CpG DNA, GM-CSF,GPI-0100, IFA, IFN-γ, IL-17, lipid A, lipopolysaccharide, Lipovant,Montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A,trehalose dimycolate or zymosan. In some embodiments, adjuvant is onethat induces a mixed type 1/type 17 immune response.

In some embodiments of the kits or compositions provided herein, the kitor composition further comprises an additional agent (e.g., ananti-cancer agent). In some embodiments, the additional agent is ananti-cancer agent selected from the group consisting of paclitaxel,cisplatin, topotecan, gemcitabine, bleomycin, etoposide, carboplatin,docetaxel, doxorubicin, topotecan, cyclophosphamide, trabectedin,olaparib, tamoxifen, letrozole and bevacizumab.

In some embodiments, the anti-cancer agent is an immune checkpointinhibitor. In some embodiments, the immune checkpoint inhibitor is aninhibitor of CTLA4, such as an anti-CTLA4 antibody (e.g., ipilimumab(BMS), tremelimumab (AstraZeneca) and/or KAHR-102 (Kahr Medical)). Insome embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-1, such as an anti-PD-1 antibody (e.g., nivolumab (BMS),pembrolizumab/lambrolizumab (Merck), pidilizumab (Curetech), AMP-224(GSK), AMP-514 (AstraZeneca), STI-A1110 (Sorrento) and/or TSR-042(Tesaro). In some embodiments, the immune checkpoint inhibitor is aninhibitor of PD-L1 and/or PD-L2, such as an anti-PD-L1 and/or ananti-PD-L2 antibody (e.g., RG-7446 (Roche), BMS-936559 (BMS), MEDI-4736(AstraZeneca), MSB-0020718C (Merck), AUR-012 (Pierre Fabre Med),STI-A1010 (Sorrento)).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 has three panels. Panel (A) is a schematic representation of fulllength AMHR2 showing the extracellular, transmembrane, and cytoplasmicdomains with a C-terminal 6×His-tagged (SEQ ID NO: 32) AMHR2-CD variant.Panel (B) depicts an SDS-PAGE gel stained with Coomassie blue showingexpression of AMHR2-CD in non-induced, IPTG-induced, and Ni-NTA affinitypurified AMHR2-CD. Panel (C) depicts an anti-His Western blot of anSDS-PAGE gel showing expression of AMHR2-CD in non-induced,IPTG-induced, and two doses of Ni-NTA affinity purified AMHR2-CD.

FIG. 2 has six panels and shows data resulting from the immunization offemale C57BL/6 mice with AMHR2-CD in CFA, and LNC or splenocytes werecultured in vitro for assessment of proliferation and cytokineproduction. Panel (A) demonstrates that 10 day primed LNC showed markedantigen-specific recall proliferative responses to AMHR2-CD over severallogs of antigen concentration. Panel (B) shows that a response toAMHR2-CD was elicited by CD4⁺ T cells but not by CD8⁺ T cells purifiedby magnetic bead separation. Panel (C) shows proliferative responses toAMHR2-CD were markedly inhibited in the presence of CD4 antibody but notin the presence of CD8 or isotype control antibodies. Panel (D) showsthat four weeks after immunization, splenocytes were reactivated withimmunogen and ELISA analysis of 72 hour culture supernatants showed thatrecall responses to AMHR2-CD involved a proinflammatory phenotype withelevated production of IFNγ and minimal production of IL-2, IL-4, andIL-5. Panel (E) shows splenocyte production of IFNγ was elicited frompurified CD4⁺ T cells but not from purified CD8⁺ T cells. Panel (F)shows that two months after immunization, serum levels ofAMHR2-CD-specific IgG were detectable even at titers over a 1:50,000dilution. PBS was substituted for diluted sera in the PBS control. Errorbars show ±SD.

FIG. 3 has three panels and shows data related to benign transientovarian inflammation following AMHR2-CD immunization. Panel (A) showsrelative ovarian IFNγ gene expression was elevated 4 weeks afterimmunization with AMHR2-CD but not after immunization with CFA alone. Ateight weeks after immunization, relative ovarian IFNγ gene expressionwas similar in both immunized groups of mice. Panel (B) shows the lowlevel transient expression of IFNγ in ovaries of AMHR2-CD immunized micewas not associated with any detectable effect on ovarian function asdetermined by assessing fertility defined by pup production over foursequential mating cycles in female C57BL/6 mice immunized with AMHR2-CDand control mice immunized with CFA alone. Panel (C) shows that AMHR2gene expression was confined to ovaries and ID8 ovarian tumor cells andwas not detected in normal uterus, stomach, spleen, heart, lung, kidney,and liver. Error bars show ±SD.

FIG. 4 has seven panels and shows data related to the inhibition oftumor growth in mice immunized with AMHR2-CD. As depicted in Figures(A), (B) and (C), respectively, ID8 tumor growth was inhibited in miceprophylactically vaccinated 5 days, 7 days or 1 day prior to inoculationof tumor cells. Panel (D) shows that AMHR2-CD vaccination resulted in asignificantly decreased overall tumor load as measured by final tumorweight at termination of experiments in mice vaccinated 7 days and 1 dayprior to ID8 inoculation. Panel (E) shows that therapeutic vaccinationwith AMHR2-CD 60 days after inoculation of ID8 tumors significantlyinhibited the growth of established, palpable, growing ID8 tumors. Panel(F) shows that prophylactic vaccination of female TgM1SIIR-TAgtransgenic mice at 6-7 weeks of age with AMHR2-CD resulted in a highlysignificant inhibition in growth of autochthonous EOC. Panel (G) showsthat prophylactic AMHR2-CD vaccination of female TgM1SIIR-TAg transgenicmice at 6-7 weeks of age resulted in a highly significant 41.7% meanincreased OS compared to control mice vaccinated with CFA alone.Asterisks indicate statistical significance. Error bars show ±SD.

FIG. 5 has three panels and shows data related to tumor analysis. Inpanel (A), arrows indicate infiltration of CD3⁺ T cells in an ID8 tumorfrom AMHR2-CD vaccinated mice in lower resolution (top) and higherresolution (bottom) images. Inflammatory infiltrates of CD3⁺ T cellswere never observed in control mice vaccinated with CFA alone. Panel (B)depicts a flow cytometry analysis of TILs gated on the CD3⁺ T cellpopulation showed a pronounced increase in percentages of CD4+ T cellsbut not CD8+ T cells in tumor infiltrates from mice vaccinated withAMHR2-CD compared to control mice immunized with CFA alone. Data shownare representative of three experiments yielding similar results. Panel(C) shows that tumors from AMHR2-CD immunized mice consistently showedincreased relative gene expression for CD4, IFNγ, TNFα, and IL-2 but notfor CD8. Asterisks indicate statistical significance. Error bars show±SD.

FIG. 6 has five panels and shows data related to the passive transfer ofimmune protection against tumor growth with CD4⁺ T cells. Recipient micewere inoculated with ID8 tumor cells on the day after cell transfer.Growth of ID8 tumors was inhibited in mice transferred withAMHR2-CD-specific LNCs (panel (A)) and splenocytes (panel (B)). Panel(C) shows that at 190 days after splenocyte transfer and inoculation,mean tumor weights were lower in recipients of AMHR2-CD-specificsplenocytes compared to recipients of OVA-specific splenocytes. Panels(D) and (E) respectively show that transfer of purifiedAMHR2-CD-specific CD4⁺ T cells but not B220⁺ B cells inhibited ID8 tumorgrowth. Asterisks indicate statistical significance. Error bars show±SE.

FIG. 7 shows AMHR2 gene expression in normal human tissues. Twenty-fiveother normal human tissues were also examined and showed no expressionof AMHR2 above background levels.

FIG. 8 has five panels and shows AMHR2 gene expression in human tissues.Panel (A) shows that the extracellular domain of AMHR2 is expressed inthe ovary but not in other AMHR2-expressing tissues. Panel (B) showsthat using primer pairs specific for AMHR2-CD and AMHR2-ED, qRT-PCRanalysis of human tissues confirms findings from the Human Protein Atlasby indicating that AMHR2-ED is expressed exclusively in the human ovaryand is not expressed in the human adrenal gland or pancreas. Panel (C)shows ovarian expression of human AMHR2 declines with age and thisdecline is particularly evident with respect to AMHR2-ED which isexpressed at significantly lower levels in the postmenopausal ovary(mean age, 64 years) compared to the premenopausal ovary (mean age, 31years). A similar significant decline in AMHR2-ED gene expression occursin older mouse ovaries (9 months of age). Panel (D) shows the declinewith age in ovarian expression of AMHR2 stands in contrast to the highlevel of gene expression of both AMHR2-CD and AMHR2-ED domains in humanEOC. Panel (E) shows that this high AMHR2 gene expression in human EOCis accompanied by corresponding high levels of detectable AMHR2 proteindetermined by Western blot analysis on all 16 human EOC tumors examinedusing a polyclonal antibody specific for AMHR2-ED with β-actinimmunostaining serving as endogenous control. In all cases, error barsindicate ±SD, and asterisks indicate significance.

FIG. 9 has two panels and shows expression of the AMHR2 extracellulardomain in human EOCs. Panel (A) shows that using primer pairs specificfor AMHR2-ED, quantitative real-time RT-PCR analysis shows expression ofAMHR2-ED in normal human ovaries (n=7) but not in normal human adrenalglands (n=3). Panel (B) shows a western blot analysis using a polyclonalantibody specific for the extracellular domain of AMHR2 shows detectionof AMHR2-ED protein in all five human EOCs examined. The full-lengthprotein was detected in only one of the five examined tumors suggestingthat AMHR2 signaling may be altered in many EOCs due to possibledeletions of regions of the cytoplasmic kinase domain. Antibody specificfor 13-Actin was used as a control.

FIG. 10 has eight panels. Panel (A) is a schematic representation offull-length mouse AMHR2 (upper) as well as the 140 amino acid AMHR2-EDHIS-tagged truncated variant selected for production as a recombinantimmunogen. “6×-His” is disclosed as SEQ ID NO: 32. Panel (B) shows ananti-His Western blot of SDS-PAGE gel showing expression of Ni-NTAaffinity purified AMHR2-ED at two loaded doses (left) and Coomasie Bluestain of same (right). Panel (C) shows one month after AMHR2-EDvaccination of eight week old female C57BL/6 mice; ELISPOT analysisshowed elevated splenocyte frequencies of antigen-specific T cellsproducing IFNγ and IL-17 but not IL-5. Panel (D) shows IFNγ and panel(E) shows IL-17 proinflammatory response to AMHR2-ED was duepredominantly to responding CD4+ T cells but also incorporated anoticeable response from CD8+ T cells. Panel (F) shows AMHR2-EDvaccination induced a substantial serum IgG antibody response againstAMHR2-ED that was detectable at dilutions up to 1/50,000 and panel (G)shows predominantly involved the IgG1 and IgG2b isotypes. Panel (H)shows that the immunogenicity of AMHR2-ED was not confined to C57BL/6mice since female mice representing three other divergent H-2 haplotypesshowed high antigen-specific frequencies of IFNγ-producing T cells. Inall cases, error bars indicate ±SD.

FIG. 11 has five panels and shows that AMHR2-ED is highly immunogenic.Panel (A) shows that splenocytes from AMHR2-ED immunized mice showedantigen-specific recall proliferative responses to AMHR2-ED but not torecombinant mouse 3-casein, an irrelevant control antigen generated andpurified in a manner similar to AMHR2-ED. Panel (B) shows thatsplenocytes from CFA immunized mice were unresponsive to both AMHR2-EDand β-casein. Panel (C) depicts an ELISA analysis of culturesupernatants, which shows AMHR2-ED activated production of high levelsof the proinflammatory cytokines, IFNγ and IL-17, and minimal productionof the type-2 regulatory cytokine, IL-5. Panel (D) shows thatsplenocytes from AMHR2-ED immunized mice demonstrate significantly highfrequencies of type-1 (˜1/4,000 lymphocytes; p<0.0001) and type-17(˜1/20,000 lymphocytes; p<0.02) proinflammatory T cells but minimalfrequencies of type-2 regulatory T cells expressing IL-5. Panel (E)shows that four months after immunization with AMHR2-EC, serum titersfor AMHR2-ED specific IgG were significantly detectable at titersexceeding 1/50,000 dilutions (p<0.001).

FIG. 12 has five panels and shows that AMHR2-ED activates apredominantly CD4⁺ T cell response. Splenocytes and purified CD4+ butnot CD8+ T cells isolated one month after immunization of TgM1SIIR-TAgtransgenic female mice with AMHR2-ED showed a prominent antigen-specificinduction of type-1 (panel (A)) and type-17 (panel (B)) proinflammatoryT cells. Immunohistochemical analysis of autochthonous EOC taken from 7month old female TgMISIIR-Tag mice that were immunized at 6 weeks of agewith AMHR2-ED showed predominant infiltration of CD3⁺ T cells (panel(C)) and CD4⁺ T cells (panel (D)), but not CD8⁺ T cells (panel (E)).

FIG. 13 has three panels and shows transient ovarian inflammationfollowing immunization with AMHR2-ED. Real-time RT-PCR analysis ofovaries taken four weeks (panel (A)) but not 8 weeks (panel (B))following immunization with AMHR2-ED showed expression of theinflammatory cytokine IL-10. IFNγ expression was not elevated at timepoint following immunization. At four weeks (panel (C)) after AMHR2-EDvaccination of 9 month old C57BL/6 female mice, qRT-PCR analysis showedno elevated ovarian gene expression for either IL-1β or IFNγ.

FIG. 14 has two panels and shows that mouse fertility was unaffected byAMHR2-ED immunization. No significant differences in mean number of pupsper litter (panel (A)) or mean pup birth weights (panel (B)) weredetected between mice immunized with AMHR2-ED in CFA or control miceimmunized with CFA alone.

FIG. 15 has eight panels and shows that AMHR2-ED vaccination inhibitsgrowth of authochthonous and transplantable ovarian tumors. Panel (A)shows that prophylactic AMHR2-ED vaccination of female TgM1SIIR-TAgtransgenic mice at 6-7 weeks of age resulted in a highly significantinhibition in growth of autochthonous EOC (p<0.0001). Panel (B) showsthat prophylactic AMHR2-ED vaccination of female TgM1SIIR-TAg transgenicmice at 6-7 weeks of age resulted in a 42% increased overall survivalcompared to control mice vaccinated with CFA alone (mean 193.7±34.5 daysvs. mean 135±13.89 days). Similar significant inhibition in growth oftransplantable TgMISIIR EOC tumors occurred in mice vaccinated either 7days panel (C) or 15 days panel (D) prior to inoculation with 3×10⁶mouse ovarian carcinoma (MOVCAR) cells (p<0.001). Panel (E) showsprophylactic AMHR2-ED vaccination of 6-7 week old TgMISIIR-TAg (DR26)transgenic mice significantly delayed the appearance and growth ofautochthonous EOC tumors. Panel (F) shows the inhibition in growth ofautochthonous tumors resulted in a highly significant 42% increasedoverall survival. Panel (G) shows AMHR2-ED vaccination was alsoeffective in providing significant immunotherapy against EOC inTgMISIIR-TAg (DR26) transgenic mice with established autochthonoustumors. Panel (H) shows immunohistochemical analysis of autochthonousEOC tumors from TgMISIIR-TAg (DR26) mice vaccinated with AMHR2-EDconsistently showed prominent infiltrations of CD3+ T cells (upper leftpanel) and CD4+ T cells (upper middle panel) with occasional CD8+ Tcells (upper right panel) indicated by arrows. Correspondingimmunostained EOC tumors from control mice vaccinated with CFA aloneconsistently failed to show detectable T cell infiltrates (lowerpanels). In all cases, error bars indicate ±SD, and asterisks indicatesignificance.

FIG. 16 has two panels and shows inhibition of EOC tumor growthfollowing transfer of AMHR2-ED primed CD4⁺ T cells and B220⁺ B cells.Inhibition of growth of MOVCAR EOCs occurred following transfer of CD4⁺T cells (panel (A)) or B220⁺ B cells (panel (B)) from mice immunizedwith AMHR2-ED. Asterisks indicate significance.

FIG. 17 has two panels and shows increased overall survival followingtransfer of AMHR2-ED primed CD4⁺ T cells and B220⁺ B cells. Increasedoverall survival in MOVCAR tumor bearing mice that received CD4+ T cellspanel (A) or B220+ B cells panel (B) from mice immunized with AMHR2-ED.

FIG. 18 shows inhibition of tumor growth and enhancement of overallsurvival following transfer of AMHR2-ED primed CD4⁺ T cells and sera.Growth inhibition of ID8-VEGF EOC tumors occurred in mice receiving CD4+T cells (upper left panel) or sera (lower left panel) from miceimmunized with AMHR2-ED. Enhanced overall survival occurred in mice thatreceived CD4⁺ T cells (upper right panel) or sera (lower right panel)from mice immunized with AMHR2-ED.

FIG. 19 is the amino acid sequence of the longest of the human AMHR2-CDprotein variant (SEQ ID NO: 1). The extracellular domain (AMHR2-ED) isindicated in bold and the cytoplasmic domain (AMHR2-CD) is indicated initalics.

FIG. 20 has six panels and shows the passive transfer of tumor immunitywith CD4+ T Cells, B220+ B Cells, and Purified IgG. Panel (A) shows thetransfer of AMHR2-ED primed CD4+ T cells into TgMISIIR-TAg (low) femalemice one day prior to MOVCAR inoculation resulted in significantinhibition of tumor growth and panel (B) shows enhanced overall survivalcompared to mice receiving ovalbumin primed CD4+ T cells. Panel (C)shows transfer of AMHR2-ED primed B220+ B cells into TgMISIIR-TAg (low)female mice one day prior to MOVCAR inoculation resulted in significantinhibition of tumor growth and panel (D) shows enhanced overall survivalcompared to mice receiving B220+ B cells from ovalbumin immunized mice.Panel (E) shows transfer of affinity purified IgG from AMHR2-EDimmunized mice into TgMISIIR-TAg (low) female mice one day prior toMOVCAR inoculation resulted in significant inhibition of tumor growthand panel (F) shows enhanced overall survival compared to mice receivingaffinity purified IgG from ovalbumin immunized mice. In all cases, errorbars indicate ±SD, and asterisks indicate significance.

DETAILED DESCRIPTION General

Provided herein are methods, kits and compositions for the treatmentand/or prevention of ovarian cancer through the induction of an immuneresponse against Anti-Mullerian Hormone Receptor, Type II (AMHR2).

AMHR2 is a serine/threonine kinase receptor homologous to type IIreceptors of the transforming growth factor beta (TGFβ) family. Thehuman AMHR2 gene contains 11 exons with seven known alternativelyspliced variants producing three known coded proteins, one additionalvariant with protein coding features, and three non-coding transcriptswith no open reading frames. In adult women, the longest human proteincoding transcript for a 573 amino acid long protein (SEQ ID NO: 1; FIG.19) is normally expressed only in the ovary and comprises a 127 aminoacid extracellular domain (AMHR2-ED), a 26 amino acid transmembranedomain, and a 403 amino acid cytoplasmic domain (AMHR2-CD). AMHR2signaling causes regression of the Müllerian ducts during maledevelopment and regulates oocyte development and follicle production inadult females, thereby providing substantial control of ovarian reserveand fertility. AMHR2 is expressed in the vast majority of human EOCs,including 90% of primary EOCs, 78% of borderline malignancies, 77-86% ofnon-EOC ovarian tumors, and 56% of malignant ascites from grade III-IVovarian cancers. In normal tissues, AMHR2-CD expression is predominantlyconfined to the ovaries. While some AMHR2-CD expression also occurs in asmall number of additional human tissues, AMHR2-ED is expressedexclusively in the ovary. What is more, ovarian expression of bothAMHR2-CD and AMHR2-ED is reduced in post-menopausal ovaries.

As described herein, vaccination with AMHR2 polypeptides and/or adoptivetransfer of AMHR2-primed lymphocytes provides effective immunotherapyagainst ovarian cancer without producing extensive autoimmunecomplications. Immunization of a mouse model for ovarian cancer witheither recombinant mouse AMHR2 polypeptides containing either a 399amino acid sequence of the cytoplasmic domain (AMHR2-CD) or a 140 aminoacid sequence of the extracellular domain (AMHR2-ED) resulted in aprominent proinflammatory T cell response accompanied by extremely highIgG antibody titers. Vaccination with AMHR2-CD and AMHR2-ED polypeptidesprovided significant T cell-mediated prophylaxis and therapy againstovarian cancer and mediated significant prophylaxis against thedevelopment of autochthonous ovarian cancer tumors in the mouse ovariancancer models. Moreover, the protection against tumor growth wasaccompanied by minimal autoimmune symptoms, with no detectable effectson fertility over the course of several subsequent mating cycles. Thesedata indicate that targeted vaccination against AMHR2-CD and/or AMHR2-EDprovides a safe and effective therapy against ovarian cancer.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “administering” means providing apharmaceutical agent or composition to a subject, and includes, but isnot limited to, administering by a medical professional andself-administering.

The term “immune response” refers herein to any response to an antigenor antigenic determinant by the immune system. Exemplary immuneresponses include humoral immune responses (e.g. production ofantigen-specific antibodies (neutralizing or otherwise)) andcell-mediated immune responses (e.g. lymphocyte proliferation). Type-1inflammatory immune responses are characterized by the production oftype-1 cytokines, such as IFNγ. Type-2 inflammatory immune responses arecharacterized by expression of type-2 cytokines, such as IL-4 or IL-5.Type-17 inflammatory immune responses are characterized by expression oftype-17 cytokines, and particularly IL-17. In some instances, a mixedimmune response can be generated. For example, in some instances a mixedtype-1/type-17 inflammatory immune response is generated that ischaracterized by the expression of both IFNγ and IL-17.

As used herein, “percent identity” between amino acid sequences issynonymous with “percent homology,” which can be determined using thealgorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87,2264-2268, 1990), modified by Karlin and Altschui (Proc. Natl. Acad.Sci. USA 90, 5873-5877, 1993). The noted algorithm is incorporated intothe NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215,403-410, 1990). BLAST nucleotide searches are performed with the NBLASTprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a polynucleotide described herein. BLAST protein searchesare performed with the XBLAST program, score=50, wordlength=3, to obtainamino acid sequences homologous to a reference polypeptide. To obtaingapped alignments for comparison purposes, Gapped BLAST is utilized asdescribed in Altschul et al. (Nucleic Acids Res. 25, 3389-3402, 1997).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) are used.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody.

As used herein, the term “subject” means a human or non-human animalselected for treatment or therapy.

The phrases “therapeutically-effective amount” and “effective amount” asused herein means the amount of an agent which is effective forproducing the desired therapeutic effect in at least a sub-population ofcells in a subject at a reasonable benefit/risk ratio applicable to anymedical treatment.

“Treating” a disease in a subject or “treating” a subject having adisease refers to subjecting the subject to a pharmaceutical treatment,e.g., the administration of a drug, such that at least one symptom ofthe disease is decreased or prevented from worsening.

Anti-Müllerian Hormone Receptor II (AMHR2)

In certain aspects, provided herein are provided herein are methods,kits and compositions for the treatment and/or prevention of ovariancancer through the induction of an immune response againstAnti-Mullerian Hormone Receptor, Type II (AMHR2) through theadministration of an AMHR2 polypeptide (e.g., a polypeptide containingthe AMHR2-ED or an immunogenic fragment thereof, or the AMHR2-CD or animmunogenic fragment thereof) or a nucleic acid encoding an AMHR2polypeptide.

The human AMHR2 gene contains 11 exons with seven known alternativelyspliced variants producing three known coded proteins, one additionalvariant with protein coding features, and three non-coding transcriptswith no open reading frames. In adult women, the longest human proteincoding transcript for a 573 amino acid long protein is normallyexpressed only in the ovary and comprises a 127 amino acid extracellulardomain, a 26 amino acid transmembrane domain, and a 403 amino acidcytoplasmic domain (FIG. 19). AMHR2 signaling causes regression of theMüllerian ducts during male development and regulates oocyte developmentand follicle production in adult females, thereby providing substantialcontrol of ovarian reserve and fertility. The mRNA sequences of thethree protein-coding isoforms of AMHR2 are provided at NCBI referencenumbers NM_020547.2, NM_001164690.1 and NM_001164690.1, which encode forproteins having amino acid sequences provided at NCBI reference numbersNP_065434.1, NP_001158162.1 and NP_001158163.1, respectively. Each ofthe above mRNA and protein sequences are hereby incorporated byreference.

In some embodiments, provided herein are AMHR2 polypeptides and/ornucleic acids encoding AMHR2 polypeptides. AMHR2 polypeptides arepolypeptides that include an amino acid sequence that corresponds to theamino acid sequence of an AMHR2 protein, the AMHR2-ED, the AMHR2-CD,and/or a portion of the AMHR2 amino acid sequence of sufficient lengthto elicit an AMHR2-specific immune response. In certain embodiments, theAMHR2 polypeptide also includes amino acids that do not correspond tothe amino acid sequence (e.g., a fusion protein comprising an AMHR2amino acid sequence and an amino acid sequence corresponding to anon-AMHR2 protein or polypeptide). In some embodiments, the AMHR2polypeptide only includes amino acid sequence corresponding to an AMHR2protein or fragment thereof.

In certain embodiments of the methods, compositions and kits providedherein, the AMHR2 polypeptide has an amino acid sequence that comprisesat least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 105, 110, 115, 120, 125, 130, 140, 150, 160, 170, 180, 190or 200 consecutive amino acids of an AMHR2 protein amino acid sequence.In some embodiments, the consecutive amino acids are identical to anamino acid sequence in the cytoplasmic domain of AMHR2. In someembodiments, the consecutive amino acids are identical to an amino acidsequence in the extracellular domain of AMHR2.

In certain embodiments of the methods provided herein, the AMHR2polypeptide has an amino acid sequence that consists essentially of atleast 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 105, 110, 115, 120, 125, 130, 140, 150, 160, 170, 180, 190 or200 consecutive amino acids of an AMHR2 protein amino acid sequence. Insome embodiments, the consecutive amino acids are identical to an aminoacid sequence in the cytoplasmic domain of AMHR2. In some embodiments,the consecutive amino acids are identical to an amino acid sequence inthe extracellular domain of AMHR2.

In certain embodiments of the methods provided herein, the AMHR2polypeptide has an amino acid sequence that consists of at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 140, 150, 160, 170, 180, 190 or 200consecutive amino acids of an AMHR2 protein amino acid sequence. In someembodiments, the consecutive amino acids are identical to an amino acidsequence in the cytoplasmic domain of AMHR2. In some embodiments, theconsecutive amino acids are identical to an amino acid sequence in theextracellular domain of AMHR2.

In some embodiments of the methods provided herein, the AMHR2polypeptide has an amino acid sequence that comprises 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,120, 125, 130, 140, 150, 160, 170, 180, 190 or 200 consecutive aminoacids that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 77%, 98% or 99% identical to an amino acid sequence in anAMHR2 protein. In some embodiments, the amino acid sequence is in thecytoplasmic domain of an AMHR2 protein. In some embodiments, the aminoacid sequence is in the extracellular domain of an AMHR2 protein.

In some embodiments of the methods provided herein, the AMHR2polypeptide has an amino acid sequence that consists essentially of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 140, 150, 160, 170, 180, 190 or 200consecutive amino acids that are at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 77%, 98% or 99% identical to an amino acidsequence in an AMHR2 protein. In some embodiments, the amino acidsequence is in the cytoplasmic domain of an AMHR2 protein.

In some embodiments, the amino acid sequence is in the extracellulardomain of an AMHR2 protein.

In some embodiments of the methods, compositions and kits providedherein, the AMHR2 polypeptide has an amino acid sequence that consistsof 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 140, 150, 160, 170, 180, 190 or 200consecutive amino acids that are at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 77%, 98% or 99% identical to an amino acidsequence in an AMHR2 protein. In some embodiments, the amino acidsequence is in the cytoplasmic domain of an AMHR2 protein. In someembodiments, the amino acid sequence is in the extracellular domain ofan AMHR2 protein.

In some embodiments of the methods, compositions and kits providedherein, the AMHR2 polypeptide does not comprise an amino acid sequenceidentical to the extracellular domain of AMHR2. In some embodiments, theAMHR2 polypeptide does not comprise an amino acid sequence identical tothe transmembrane domain of AMHR2. In some embodiments, the AMHR2polypeptide does not comprise an amino acid sequence identical to thecytoplasmic domain of AMHR2.

As is well-known to those skilled in the art, polypeptides havingsubstantial sequence similarities can cause identical or very similarimmune reaction in a host animal. Accordingly, in some embodiments, aderivative, equivalent, variant, fragment, or mutant of AMHR2 protein orfragment thereof can also suitable for the methods, compositions andkits provided herein.

In some embodiments, variations or derivatives of the AMHR2 polypeptidesare provided herein. The altered polypeptide may have an altered aminoacid sequence, for example by conservative substitution, yet stillelicits immune responses which react with the unaltered protein antigen,and are considered functional equivalents. As used herein, the term“conservative substitution” denotes the replacement of an amino acidresidue by another, biologically similar residue. It is well known inthe art that the amino acids within the same conservative group cantypically substitute for one another without substantially affecting thefunction of a protein. According to certain embodiments, the derivative,equivalents, variants, or mutants of the ligand-binding domain of anAMHR2 polypeptide are polypeptides that are at least 85% homologous asequence of the AMH-R2 protein or fragment thereof. In some embodiments,the homology is at least 90%, at least 95%, or at least 98%.

In some embodiments, provided herein is a nucleic acid encoding an AMHR2polypeptide described herein, such as a DNA molecule encoding an AMHR2polypeptide. In some embodiments the composition comprises an expressionvector comprising an open reading frame encoding an AMHR2 polypeptide.In some embodiments, the AMHR2 nucleic acid includes regulatory elementsnecessary for expression of the open reading frame. Such elements caninclude, for example, a promoter, an initiation codon, a stop codon, anda polyadenylation signal. In addition, enhancers can be included. Theseelements can be operably linked to a sequence that encodes the AMHR2polypeptide.

Examples of promoters include but are not limited to promoters fromSimian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, HumanImmunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR)promoter, Moloney virus, Cytomegalovirus (CMV) such as the CMV immediateearly promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) aswell as promoters from human genes such as human actin, human myosin,human hemoglobin, human muscle creatine, and human metalothionein.Examples of suitable polyadenylation signals include but are not limitedto SV40 polyadenylation signals and LTR polyadenylation signals.

In addition to the regulatory elements required for expression, otherelements may also be included in the nucleic acid molecule. Suchadditional elements include enhancers.

Enhancers include the promoters described hereinabove. Preferredenhancers/promoters include, for example, human actin, human myosin,human hemoglobin, human muscle creatine and viral enhancers such asthose from CMV, RSV and EBV.

In some embodiments, the nucleic acid can be operably incorporated in acarrier or delivery vector. Useful delivery vectors include but are notlimited to biodegradable microcapsules, immuno-stimulating complexes(ISCOMs) or liposomes, and genetically engineered attenuated livecarriers such as viruses or bacteria.

In some embodiments, the vector is a viral vector, such as lentiviruses,retroviruses, herpes viruses, adenoviruses, adeno-associated viruses,vaccinia viruses, baculoviruses, Fowl pox, AV-pox, modified vacciniaAnkara (MVA) and other recombinant viruses. For example, a vacciniavirus vector can be used to infect dendritic cells.

Pharmaceutical Compositions

In certain aspects, provided herein are pharmaceutical compositions(e.g., a vaccine composition) comprising an AMHR2 polypeptide describedherein and/or a nucleic acid encoding an AMHR2 polypeptide describedherein. In some embodiments, the composition comprises apharmaceutically acceptable carrier. In some embodiments, thecomposition includes a combination of multiple e.g., two or more) AMHR2polypeptides or nucleic acids described herein.

The pharmaceutical compositions disclosed herein may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,e.g., those targeted for buccal, sublingual, and systemic absorption,boluses, powders, granules, pastes for application to the tongue; or (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation.

Methods of preparing these formulations or compositions include the stepof bringing into association an AMHR2 polypeptide and/or nucleic aciddescribed herein with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association an agent described herein withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Pharmaceutical compositions suitable for parenteral administrationcomprise AMHR2 polypeptides and/or nucleic acids described herein incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Properfluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Regardless of the route of administration selected, the agents providedherein, which may be used in a suitable hydrated form, and/or thepharmaceutical compositions disclosed herein, are formulated intopharmaceutically-acceptable dosage forms by conventional methods knownto those of skill in the art.

In some embodiments, the pharmaceutical composition described, whenadministered to a subject, can elicit an immune response against a cellthat expresses AMHR2. Such pharmaceutical compositions can be useful asvaccine compositions for prophylactic and/or therapeutic treatment ofovarian cancer.

In some embodiments, the pharmaceutical composition further comprises aphysiologically acceptable adjuvant. In some embodiments, the adjuvantemployed provides for increased immunogenicity of the pharmaceuticalcomposition. The adjuvant can be one that provides for slow release ofantigen (e.g., the adjuvant can be a liposome), or it can be an adjuvantthat is immunogenic in its own right thereby functioning synergisticallywith antigens (i.e., antigens present in the AMHR2 polypeptide). Forexample, the adjuvant can be a known adjuvant or other substance thatpromotes antigen uptake, recruits immune system cells to the site ofadministration, or facilitates the immune activation of respondinglymphoid cells. Adjuvants include, but are not limited to,immunomodulatory molecules (e.g., cytokines), oil and water emulsions,aluminum hydroxide, glucan, dextran sulfate, iron oxide, sodiumalginate, Bacto-Adjuvant, synthetic polymers such as poly amino acidsand co-polymers of amino acids, saponin, paraffin oil, and muramyldipeptide. In some embodiments, the adjuvant is Adjuvant 65, α-GalCer,aluminum phosphate, aluminum hydroxide, calcium phosphate, j3-GlucanPeptide, CpG DNA, GM-CSF, GPI-0100, IFA, IFN-γ, IL-17, lipid A,lipopolysaccharide, Lipovant, Montanide,N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, trehalosedimycolate or zymosan. In some embodiments, adjuvant is one that inducesa mixed type 1/type 17 immune response.

In some embodiments, the adjuvant is an immunomodulatory molecule. Forexample, the immunomodulatory molecule can be a recombinant proteincytokine, chemokine, or immunostimulatory agent or nucleic acid encodingcytokines, chemokines, or immunostimulatory agents designed to enhancethe immunologic response.

Examples of immunomodulatory cytokines include interferons (e.g., IFNα,IFNβ and IFNγ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-12, IL-17 and IL-20), tumor necrosis factors(e.g., TNFα and TNFβ), erythropoietin (EPO), FLT-3 ligand, gIp10, TCA-3,MCP-1, MIF, MIP-1.alpha., MIP-1β, Rantes, macrophage colony stimulatingfactor (M-CSF), granulocyte colony stimulating factor (G-CSF), andgranulocyte-macrophage colony stimulating factor (GM-CSF), as well asfunctional fragments of any of the foregoing.

In some embodiments, an immunomodulatory chemokine that binds to achemokine receptor, i.e., a CXC, CC, C, or CX3C chemokine receptor, alsocan be included in the compositions provided here. Examples ofchemokines include, but are not limited to, Mip1α, Mip-1β, Mip-3α(Larc), Mip-3β, Rantes, Hcc-1, Mpif-1, Mpif-2, Mcp-1, Mcp-2, Mcp-3,Mcp-4, Mcp-5, Eotaxin, Tarc, Elc, 1309, IL-8, Gcp-2 Gro-α, Gro-β, Gro-γ,Nap-2, Ena-78, Gcp-2, Ip-10, Mig, I-Tac, Sdf-1, and Bca-1 (Blc), as wellas functional fragments of any of the foregoing.

In certain embodiments, compositions provided herein also comprise oneor more other agents such as, but not limited to, chemotherapeutic,immunotherapeutic, immunomodulatory and/or anti-angiogenic agents.

In some embodiments, the one or more other agents can be achemotherapeutic agent, naturally occurring or synthetic, for example asdescribed in “Cancer Chemotherapeutic Agents”, American ChemicalSociety, 1995, W. O. Foye Ed.

In one embodiment, the chemotherapeutic agent is selected from the groupconsisting of a small molecule receptor antagonists such as vatalanib,SU 11248 or AZD-6474, EGFR or HER2 antagonists such as gefitinib,erlotinib, CI-1033 or Herceptin, antibodies such as bevacizumab,cetuximab, rituximab, DNA alkylating drugs such as cisplatin,oxaliplatin or carboplatin, anthracyclines such as doxorubicin orepirubicin, an antimetabolite such as 5-FU, pemetrexed, gemcitabine orcapecitabine, a camptothecin such as irinotecan or topotecan, ananti-cancer drug such as paclitaxel or docetaxel, an epipodophyllotoxinsuch as etoposide or teniposide, a proteasome inhibitor such asbortezomib or anti-inflammatory drugs such as celecoxib or rofecoxib,optionally in form of the pharmaceutically acceptable salts, in form ofthe hydrates and/or solvates and optionally in the form of theindividual optical isomers, mixtures of the individual enantiomers orracemates thereof.

In another embodiment, the chemotherapeutic agent is selected from thegroup consisting of a small molecule VEGF receptor antagonist such asvatalanib (PTK-787/ZK222584), SU-5416, SU-6668, SU-11248, SU-14813,AZD-6474, AZD-2171, CP-547632, CEP-7055, AG-013736, IM-842 or GW-786034,a dual EGFR/HER2 antagonist such as gefitinib, erlotinib, CI-1033 orGW-2016, an EGFR antagonist such as iressa (ZD-1839), tarceva (OSI-774),PKI-166, EKB-569, HKI-272 or herceptin, an antagonist of themitogen-activated protein kinase such as BAY-43-9006 or BAY-57-9006, aquinazoline derivative such as4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-bute-n-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)quinazolineor4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-(homomorpholin-4-yl)-1-oxo-2-bu-ten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline,or a pharmaceutically acceptable salt thereof, a protein kinase receptorantagonist which is not classified under the synthetic small moleculessuch as atrasentan, rituximab, cetuximab, Avastin™ (bevacizumab),IMC-1C11, erbitux (C-225), DC-101, EMD-72000, vitaxin, imatinib, aprotein tyrosine kinase inhibitor which is a fusion protein such asVEGFtrap, an alkylating agent or a platinum compound such as melphalan,cyclophosphamide, an oxazaphosphorine, cisplatin, carboplatin,oxaliplatin, satraplatin, tetraplatin, iproplatin, mitomycin,streptozocin, carmustine (BCNU), lomustine (CCNU), busulfan, ifosfamide,streptozocin, thiotepa, chlorambucil, a nitrogen mustard such asmechlorethamine, an ethyleneimine compound, an alkylsulphonate,daunorubicin, doxorubicin (adriamycin), liposomal doxorubicin (doxil),epirubicin, idarubicin, mitoxantrone, amsacrine, dactinomycin,distamycin or a derivative thereof, netropsin, pibenzimol, mitomycin,CC-1065, a duocarmycin, mithramycin, chromomycin, olivomycin, aphtalanilide such as propamidine or stilbamidine, an anthramycin, anaziridine, a nitrosourea or a derivative thereof, a pyrimidine or purineanalogue or antagonist or an inhibitor of the nucleoside diphosphatereductase such as cytarabine, 5-fluorouracile (5-FU), pemetrexed,tegafur/uracil, uracil mustard, fludarabine, gemcitabine, capecitabine,mercaptopurine, cladribine, thioguanine, methotrexate, pentostatin,hydroxyurea, or folic acid, a phleomycin, a bleomycin or a derivative orsalt thereof, CHPP, BZPP, MTPP, BAPP, liblomycin, an acridine or aderivative thereof, a rifamycin, an actinomycin, adramycin, acamptothecin such as irinotecan (camptosar) or topotecan, an amsacrineor analogue thereof, a tricyclic carboxamide, an histonedeacetylaseinhibitor such as SAHA, MD-275, trichostatin A, CBHA, LAQ824, orvalproic acid, an anti-cancer drug from plants such as paclitaxel(taxol), docetaxel or taxotere, a vinca alkaloid such as navelbine,vinblastin, vincristin, vindesine or vinorelbine, a tropolone alkaloidsuch as colchicine or a derivative thereof, a macrolide such asmaytansine, an ansamitocin or rhizoxin, an antimitotic peptide such asphomopsin or dolastatin, an epipodophyllotoxin or a derivative ofpodophyllotoxin such as etoposide or teniposide, a steganacin, anantimitotic carbamate derivative such as combretastatin or amphetinile,procarbazine, a proteasome inhibitor such as bortezomib, an enzyme suchas asparaginase, pegylated asparaginase (pegaspargase) or athymidine-phosphorylase inhibitor, a gestagen or an estrogen such asestramustine (T-66) or megestrol, an anti-androgen such as flutamide,casodex, anandron or cyproterone acetate, an aromatase inhibitor such asaminogluthetimide, anastrozole, formestan or letrozole, a GNrH analoguesuch as leuprorelin, buserelin, goserelin or triptorelin, ananti-estrogen such as tamoxifen or its citrate salt, droloxifene,trioxifene, raloxifene or zindoxifene, a derivative of17.beta.-estradiol such as ICI 164,384 or ICI 182,780,aminoglutethimide, formestane, fadrozole, finasteride, ketoconazole, aLH-RH antagonist such as leuprolide, a steroid such as prednisone,prednisolone, methylprednisolone, dexamethasone, budenoside,fluocortolone or triamcinolone, an interferon such as interferon .beta.,an interleukin such as IL-10 or IL-12, an anti-TNF.alpha. antibody suchas etanercept, an immunomodulatory drug such as thalidomide, its R- andS-enantiomers and its derivatives, or revimid (CC-5013), a leukotrienantagonist, mitomycin C, an aziridoquinone such as BMY-42355, AZQ orEO-9, a 2-nitroimidazole such as misonidazole, NLP-1 or NLA-1, anitroacridine, a nitroquinoline, a nitropyrazoloacridine, a“dual-function” nitro aromatic such as RSU-1069 or RB-6145, CB-1954, aN-oxide of nitrogen mustard such as nitromin, a metal complex of anitrogen mustard, an anti-CD3 or anti-CD25 antibody, a toleranceinduction agent, a biphosphonate or derivative thereof such asminodronic acid or its derivatives (YM-529, Ono-5920, YH-529),zoledronic acid monohydrate, ibandronate sodium hydrate or clodronatedisodium, a nitroimidazole such as metronidazole, misonidazole,benznidazole or nimorazole, a nitroaryl compound such as RSU-1069, anitroxyl or N-oxide such as SR-4233, an halogenated pyrimidine analoguesuch as bromodeoxyuridine, iododeoxyuridine, a thiophosphate such asWR-272 1, a photo-chemically activated drug such as porfimer, photofrin,a benzoporphyrin derivative, a pheophorbide derivative, merocyanin 540(MC-540) or tin etioporpurin, an ant-template or an anti-sense RNA orDNA such as oblimersen, a non-steroidal inflammatory drug such asacetylsalicyclic acid, mesalazin, ibuprofen, naproxen, flurbiprofen,fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen,oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen,tiaprofenic acid, fluprofen, indomethacin, sulindac, tolmetin,zomepirac, nabumetone, diclofenac, fenclofenac, alclofenac, bromfenac,ibufenac, aceclofenac, acemetacin, fentiazac, clidanac, etodolac,oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid, nifluminicacid, tolfenamic acid, diflunisal, flufenisal, piroxicam, tenoxicam,lomoxicam, nimesulide, meloxicam, celecoxib, rofecoxib, or apharmaceutically acceptable salt of a non-steroidal inflammatory drug, acytotoxic antibiotic, an antibody targeting the surface molecules ofcancer cells such as apolizumab or 1D09C3, an inhibitor ofmetalloproteinases such as TIMP-1 or TIMP-2, Zinc, an inhibitor ofoncogenes such as P53 and Rb, a complex of rare earth elements such asthe heterocyclic complexes of lanthanides, a photo-chemotherapeuticagent such as PUVA, an inhibitor of the transcription factor complexESX/DRIP130/Sur-2, an inhibitor of HER-2 expression, such as the heatshock protein HSP90 modulator geldanamycin and its derivative17-allylaminogeldanamycin or 17-AAG, or a therapeutic agent selectedfrom IM-842, tetrathiomolybdate, squalamine, combrestatin A4, TNP-470,marimastat, neovastat, bicalutamide, abarelix, oregovomab, mitumomab,TLK-286, alemtuzumab, ibritumomab, temozolomide, denileukin diftitox,aldesleukin, dacarbazine, floxuridine, plicamycin, mitotane, pipobroman,plicamycin, tamoxifen and testolactone. Preferred compounds includesmall molecule VEGF receptor antagonist such as vatalanib(PTK-787/ZK222584), SU-5416, SU-6668, SU-11248, SU-14813, AZD-6474,EGFR/HER2 antagonists such as CI-1033 or GW-2016, an EGFR antagonistsuch as iressa (gefitinib, ZD-1839), tarceva (erlotinib, OSI-774),PKI-166, EKB-569, HKI-272 or herceptin, an antagonist of themitogen-activated protein kinase such as BAY-43-9006 or BAY-57-9006,atrasentan, rituximab, cetuximab, Avastin™ (bevacizumab), IMC-1C11,erbitux (C-225), DC-101, EMD-72000, vitaxin, imatinib, an alkylatingagent or a platinum compound such as melphalan, cyclophosphamide,cisplatin, carboplatin, oxaliplatin, satraplatin, daunorubicin,doxorubicin (adriamycin), liposomal doxorubicin (doxil), epirubicin,idarubicin, a pyrimidine or purine analogue or antagonist or aninhibitor of the nucleoside diphosphate reductase such as cytarabine,5-fluorouracile (5-FU), pemetrexed, tegafur/uracil, gemcitabine,capecitabine, mercaptopurine, methotrexate, an anti-cancer drug such aspaclitaxel (taxol) or docetaxel, a vinca alkaloid such as navelbine,vinblastin, vincristin, vindesine or vinorelbine, an antimitotic peptidesuch as dolastatin, an epipodophyllotoxin or a derivative ofpodophyllotoxin such as etoposide or teniposide, a non-steroidalinflammatory drug such as meloxicam, celecoxib, rofecoxib, an antibodytargeting the surface molecules of cancer cells such as apolizumab orID09C3 or the heat shock protein HSP90 modulator geldanamycin and itsderivative 17-allylaminogeldanamycin or 17-AAG.

In another embodiment, the chemotherapeutic agent is selected from thegroup consisting of compounds interacting with or binding tubulin,synthetic small molecule VEGF receptor antagonists, small moleculegrowth factor receptor antagonists, inhibitors of the EGF receptorand/or VEGF receptor and/or integrin receptors or any other proteintyrosine kinase receptors which are not classified under the syntheticsmall-molecules, inhibitors directed to EGF receptor and/or VEGFreceptor and/or integrin receptors or any other protein tyrosine kinasereceptors, which are fusion proteins, compounds which interact withnucleic acids, and which are classified as alkylating agents or platinumcompounds, compounds which interact with nucleic acids and which areclassified as anthracyclines, as DNA intercalators or as DNAcross-linking agents, including DNA minor-groove binding compounds,anti-metabolites, naturally occurring, semi-synthetic or syntheticbleomycin type antibiotics, inhibitors of DNA transcribing enzymes, andespecially the topoisomerase I or topoisomerase II inhibitors, chromatinmodifying agents, mitosis inhibitors, anti-mitotic agents, cell-cycleinhibitors, proteasome inhibitors, enzymes, hormones, hormoneantagonists, hormone inhibitors, inhibitors of steroid biosynthesis,steroids, cytokines, hypoxia-selective cytotoxins, inhibitors ofcytokines, lymphokines, antibodies directed against cytokines, oral andparenteral tolerance induction agents, supportive agents, chemicalradiation sensitizers and protectors, photo-chemically activated drugs,synthetic poly- or oligonucleotides, optionally modified or conjugated,non-steroidal anti-inflammatory drugs, cytotoxic antibiotics, antibodiestargeting the surface molecules of cancer cells, antibodies targetinggrowth factors or their receptors, inhibitors of metalloproteinases,metals, inhibitors of oncogenes, inhibitors of gene transcription or ofRNA translation or protein expression, complexes of rare earth elements,and photo-chemotherapeutic agents.

In other embodiments, the chemotherapeutic agent is selected from thegroup consisting of paclitaxel (taxol), docetaxel, a vinca alkaloid suchas navelbine, vinblastin, vincristin, vindesine or vinorelbine, analkylating agent or a platinum compound such as melphalan,cyclophosphamide, an oxazaphosphorine, cisplatin, carboplatin,oxaliplatin, satraplatin, tetraplatin, iproplatin, mitomycin,streptozocin, carmustine (BCNU), lomustine (CCNU), busulfan, ifosfamide,streptozocin, thiotepa, chlorambucil, a nitrogen mustard such asmechlorethamine, an immunomodulatory drug such as thalidomide, its R-and S-enantiomers and its derivatives, or revimid (CC-5013)), anethyleneimine compound, an alkylsulphonate, daunorubicin, doxorubicin(adriamycin), liposomal doxorubicin (doxil), epirubicin, idarubicin,mitoxantrone, amsacrine, dactinomycin, distamycin or a derivativethereof, netropsin, pibenzimol, mitomycin, CC-1065, a duocarmycin,mithramycin, chromomycin, olivomycin, a phtalanilide such as propamidineor stilbamidine, an anthramycin, an aziridine, a nitrosourea or aderivative thereof, a pyrimidine or purine analogue or antagonist or aninhibitor of the nucleoside diphosphate reductase such as cytarabine,5-fluorouracile (5-FU), uracil mustard, fludarabine, gemcitabine,capecitabine, mercaptopurine, cladribine, thioguanine, methotrexate,pentostatin, hydroxyurea, or folic acid, an acridine or a derivativethereof, a rifamycin, an actinomycin, adramycin, a camptothecin such asirinotecan (camptosar) or topotecan, an amsacrine or analogue thereof, atricyclic carboxamide, an histonedeacetylase inhibitor such as SAHA,MD-275, trichostatin A, CBHA, LAQ824, or valproic acid, a proteasomeinhibitor such as bortezomib, a small molecule VEGF receptor antagonistsuch as vatalanib (PTK-787/ZK222584), SU-5416, SU-6668, SU-11248,SU-14813, AZD-6474, AZD-2171, CP-547632, CEP-7055, AG-013736, IM-842 orGW-786034, an antagonist of the mitogen-activated protein kinase such asBAY-43-9006 or BAY-57-9006, a dual EGFR/HER2 antagonist such asgefitinib, erlotinib, CI-1033 or GW-2016, an EGFR antagonist such asiressa (ZD-1839), tarceva (OSI-774), PKI-166, EKB-569, HKI-272 orherceptin, a quinazoline derivative such as4-[(3-chloro-4-fluorophenyl)amino]-6-{[-4-(N,N-dimethylamino)-1-oxo-2-but-1-en-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazolineor4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-(homomorpholin-4-yl)-1-oxo-2-bu-ten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline,or a pharmaceutically acceptable salt thereof, an inhibitor of thetranscription factor complex ESX/DRIP130/Sur-2, an inhibitor of HER-2expression, such as the heat shock protein HSP90 modulator geldanamycinand its derivative 17-allylaminogeldanamycin or 17-AAG, a protein kinasereceptor antagonist which is not classified under the synthetic smallmolecules such as atrasentan, rituximab, cetuximab, Avastin™(bevacizumab), IMC-1C11, erbitux (C-225), DC-101, EMD-72000, vitaxin,imatinib, and an antibody targeting the surface molecules of cancercells such as apolizumab or 1D09C3.

In some embodiments, the anti-cancer agent is an immune checkpointinhibitor. In some embodiments, the immune checkpoint inhibitor is aninhibitor of CTLA4, such as an anti-CTLA4 antibody (e.g., ipilimumab(BMS), tremelimumab (AstraZeneca) and/or KAHR-102 (Kahr Medical)). Insome embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-1, such as an anti-PD-1 antibody (e.g., nivolumab (BMS),pembrolizumab/lambrolizumab (Merck), pidilizumab (Curetech), AMP-224(GSK), AMP-514 (AstraZeneca), STI-A1110 (Sorrento) and/or TSR-042(Tesaro). In some embodiments, the immune checkpoint inhibitor is aninhibitor of PD-L1 and/or PD-L2, such as an anti-PD-L1 and/or ananti-PD-L2 antibody (e.g., RG-7446 (Roche), BMS-936559 (BMS), MEDI-4736(AstraZeneca), MSB-0020718C (Merck), AUR-012 (Pierre Fabre Med),STI-A1010 (Sorrento)).

In some embodiments, the composition comprises a nucleic acid encodingan AMHR2 polypeptide described herein, such as a DNA molecule encodingan AMHR2 polypeptide. In some embodiments the composition comprises anexpression vector comprising an open reading frame encoding an AMHR2polypeptide.

When taken up by a cell (e.g., muscle cell, an antigen-presenting cell(APC) such as a dendritic cell, macrophage, etc.), a DNA molecule can bepresent in the cell as an extrachromosomal molecule and/or can integrateinto the chromosome. DNA can be introduced into cells in the form of aplasmid which can remain as separate genetic material. Alternatively,linear DNAs that can integrate into the chromosome can be introducedinto the cell. Optionally, when introducing DNA into a cell, reagentswhich promote DNA integration into chromosomes can be added.

Therapeutic Methods

In certain aspects, provided herein are methods for treating orpreventing ovarian cancer and/or for inducing an immune response againstan ovarian cancer tumor or AMHR2. In certain embodiments, the methodcomprises administering to a subject a pharmaceutical compositiondescribed herein. In some embodiments, the ovarian cancer tumor is aprimary tumor. In some embodiments, the ovarian cancer tumor is ametastatic tumor. In some embodiments, the ovarian cancer tumor is anepithelial ovarian cancer (EOC) tumor. In some embodiments, the ovariancancer tumor expresses AMHR2.

The methods described herein can be used to treat any subject in needthereof. As used herein, a “subject in need thereof” includes anysubject who has ovarian cancer, who has had ovarian cancer and/or who ispredisposed to ovarian cancer. For example, in some embodiments, thesubject has an ovarian cancer tumor (e.g., an ovarian cancer tumorexpressing AMHR2). In some embodiments, the subject has undergonesurgery to remove at least part of an ovarian cancer tumor. In someembodiments, the subject is predisposed to ovarian cancer due to havinga BRCA1 or BRCA2 mutation in her genome that predisposes the subject toovarian cancer. In some embodiments, the subject has a family history ofovarian cancer.

The pharmaceutical compositions disclosed herein may be delivered by anysuitable route of administration, including orally and parenterally. Incertain embodiments the pharmaceutical compositions are deliveredgenerally (e.g., via oral or parenteral administration).

The dosage of the subject agent may be determined by reference to theplasma concentrations of the agent. For example, the maximum plasmaconcentration (Cmax) and the area under the plasma concentration-timecurve from time 0 to infinity (AUC (0-4)) may be used. Dosages includethose that produce the above values for Cmax and AUC (0-4) and otherdosages resulting in larger or smaller values for those parameters.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular agent employed, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldprescribe and/or administer doses of the agents employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of an agent described herein will bethat amount of the agent which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above.

In one aspect, provided herein is a method of eliciting in a subject animmune response to a cell that expresses AMHR2 (e.g., an ovarian cancertumor cell). The method comprises: administering to the subject apharmaceutical composition described herein, wherein thepharmaceutically acceptable composition, when administered to thesubject, elicits an immune response to the cell that expresses AMHR2.

Generally, the immune response can include a humoral immune response, acell-mediated immune response, or both.

A humoral response can be determined by a standard immunoassay forantibody levels in a serum sample from the subject receiving thepharmaceutical composition. A cellular immune response is a responsethat involves T cells and can be determined in vitro or in vivo. Forexample, a general cellular immune response can be determined as the Tcell proliferative activity in cells (e.g., peripheral blood leukocytes(PBLs)) sampled from the subject at a suitable time following theadministering of a pharmaceutically acceptable composition. Followingincubation of e.g., PBMCs with a stimulator for an appropriate period,[³H]thymidine incorporation can be determined. The subset of T cellsthat is proliferating can be determined using flow cytometry.

In certain aspects, the methods provided herein include administering toboth human and non-human mammals. Veterinary applications also arecontemplated. In some embodiments, the subject can be any living femaleorganism in which an immune response can be elicited. Examples ofsubjects include, without limitation, humans, livestock, dogs, cats,mice, rats, and transgenic species thereof.

In certain embodiments, the subject has a history of ovarian cancer andhas been administered another mode of therapy. The other therapy mayhave included e.g., surgical resection, radiotherapy, chemotherapy, andother modes of immunotherapy whereby as a result of the other therapy,the subject presents no clinically measurable tumor. However, thesubject can be one determined to be at risk for recurrence orprogression of the cancer, either near the original tumor site, or bymetastases. Such subjects can be further categorized as high-risk andlow-risk subjects. The subdivision can be made on the basis of featuresobserved before or after the initial treatment. These features are knownin the clinical arts, and are suitably defined for each differentcancer. Features typical of high risk subgroups are those in which thetumor has invaded neighboring tissues, or which show involvement oflymph nodes. Thus, for example, a pharmaceutical composition describedherein can be administered to the subject to elicit an anti-cancerresponse primarily as a prophylactic measure against recurrence.

In some embodiments, the pharmaceutical composition can be administeredat any time that is appropriate. For example, the administering can beconducted before or during traditional therapy of a subject having anovarian cancer tumor, and continued after the tumor becomes clinicallyundetectable. The administering also can be continued in a subjectshowing signs of recurrence.

In some embodiments, the pharmaceutical composition can be administeredin a therapeutically or a prophylactically effective amount.Administering the pharmaceutical composition to the subject can becarried out using known procedures, and at dosages and for periods oftime sufficient to achieve a desired effect.

In some embodiments, the pharmaceutical composition can be administeredto the subject at any suitable site, for example a site that is distalto or proximal to a primary tumor. The route of administering can beparenteral, intramuscular, subcutaneous, intradermal, intraperitoneal,intranasal, intravenous (including via an indwelling catheter), via anafferent lymph vessel, or by any other route suitable in view of theneoplastic disease being treated and the subject's condition.Preferably, the dose will be administered in an amount and for a periodof time effective in bringing about a desired response, be it elicitingthe immune response or the prophylactic or therapeutic treatment of theneoplastic disease and/or symptoms associated therewith.

The pharmaceutically acceptable composition can be given subsequent to,preceding, or contemporaneously with other therapies including therapiesthat also elicit an immune response in the subject. For example, thesubject may previously or concurrently be treated by chemotherapy,radiation therapy, and other forms of immunotherapy, such othertherapies preferably provided in such a way so as not to interfere withthe immunogenicity of the compositions described herein.

Administering can be properly timed by the care giver (e.g., physician,veterinarian), and can depend on the clinical condition of the subject,the objectives of administering, and/or other therapies also beingcontemplated or administered. In some embodiments, an initial dose canbe administered, and the subject monitored for an immunological and/orclinical response. Suitable means of immunological monitoring includeusing patient's peripheral blood lymphocyte (PBL) as responders andneoplastic cells as stimulators. An immunological reaction also can bedetermined by a delayed inflammatory response at the site ofadministering. One or more doses subsequent to the initial dose can begiven as appropriate, typically on a monthly, semimonthly, or preferablya weekly basis, until the desired effect is achieved. Thereafter,additional booster or maintenance doses can be given as required,particularly when the immunological or clinical benefit appears tosubside.

Cell Therapy

In certain aspects, an AMHR2 polypeptide described herein, or a nucleicacid encoding such an AMHR2 polypeptide, can be used in compositions andmethods for providing AMHR2-primed, antigen-presenting cells, and/orAMHR2-specific lymphocytes generated with these antigen-presentingcells. In some embodiments, such antigen-presenting cells and/orlymphocytes are used in the treatment or prevention of ovarian cancer.

In some aspects, provided herein are methods for making AMHR2-primed,antigen-presenting cells by contacting antigen-presenting cells with anAMHR2 polypeptide described herein, or nucleic acids encoding the atleast one AMHR2 polypeptide, in vitro under a condition sufficient forthe at least one AMHR2 polypeptide to be presented by theantigen-presenting cells.

In some embodiments, the AMHR2 polypeptide, or nucleic acid encoding theAMHR2 polypeptide, can be contacted with a homogenous, substantiallyhomogenous, or heterogeneous composition comprising antigen-presentingcells. For example, the composition can include but is not limited towhole blood, fresh blood, or fractions thereof such as, but not limitedto, peripheral blood mononuclear cells, buffy coat fractions of wholeblood, packed red cells, irradiated blood, dendritic cells, monocytes,macrophages, neutrophils, lymphocytes, natural killer cells, and naturalkiller T cells. If, optionally, precursors of antigen-presenting cellsare used, the precursors can be cultured under suitable cultureconditions sufficient to differentiate the precursors intoantigen-presenting cells. In some embodiments, the antigen-presentingcells (or precursors thereof) are selected from monocytes, macrophages,cells of myeloid lineage, B cells, dendritic cells, or Langerhans cells.

The amount of the AMHR2 polypeptide, or nucleic acid encoding the AMHR2polypeptide, to be placed in contact with antigen-presenting cells canbe determined by one of ordinary skill in the art by routineexperimentation. Generally, antigen-presenting cells are contacted withthe AMHR2 polypeptide, or nucleic acid encoding the AMHR2 polypeptide,for a period of time sufficient for cells to present the processed formsof the antigens for the modulation of T cells. In one embodiment,antigen-presenting cells are incubated in the presence of the AMHR2polypeptide, or nucleic acid encoding the AMHR2 polypeptide, for lessthan about a week, illustratively, for about 1 minute to about 48 hours,about 2 minutes to about 36 hours, about 3 minutes to about 24 hours,about 4 minutes to about 12 hours, about 6 minutes to about 8 hours,about 8 minutes to about 6 hours, about 10 minutes to about 5 hours,about 15 minutes to about 4 hours, about 20 minutes to about 3 hours,about 30 minutes to about 2 hours, and about 40 minutes to about 1 hour.The time and amount of the AMHR2 polypeptide, or nucleic acid encodingthe AMHR2 polypeptide, necessary for the antigen presenting cells toprocess and present the antigens can be determined, for example usingpulse-chase methods wherein contact is followed by a washout period andexposure to a read-out system e.g., antigen reactive T cells.

In certain embodiments, any appropriate method for delivery of antigensto the endogenous processing pathway of the antigen-presenting cells canbe used. Such methods include but are not limited to, methods involvingpH-sensitive liposomes, coupling of antigens to adjuvants, apoptoticcell delivery, pulsing cells onto dendritic cells, deliveringrecombinant chimeric virus-like particles (VLPs) comprising antigen tothe MHC class I processing pathway of a dendritic cell line.

In one embodiment, solubilized AMHR2 polypeptide is incubated withantigen-presenting cells. In some embodiments, the AMHR2 polypeptide canbe coupled to a cytolysin to enhance the transfer of the antigens intothe cytosol of an antigen-presenting cell for delivery to the MHC classI pathway. Exemplary cytolysins include saponin compounds such assaponin-containing Immune Stimulating Complexes (ISCOMS), pore-formingtoxins (e.g., an alpha-toxin), and natural cytolysins of gram-positivebacteria such as listeriolysin O (LLO), streptolysin O (SLO), andperfringolysin O (PFO).

In some embodiments, antigen-presenting cells, such as dendritic cellsand macrophage, can be isolated according to methods known in the artand transfected with polynucleotides by methods known in the art forintroducing a nucleic acid encoding the AMHR2 polypeptide into theantigen-presenting cell. Transfection reagents and methods are known inthe art and commercially available. For example, RNA encoding AMHR2polypeptide can be provided in a suitable medium and combined with alipid (e.g., a cationic lipid) prior to contact with antigen-presentingcells. Non-limiting examples of such lipids include LIPOFECTIN™ andLIPOFECTAMINE™. The resulting polynucleotide-lipid complex can then becontacted with antigen-presenting cells. Alternatively, thepolynucleotide can be introduced into antigen-presenting cells usingtechniques such as electroporation or calcium phosphate transfection.The polynucleotide-loaded antigen-presenting cells can then be used tostimulate T lymphocyte (e.g., cytotoxic T lymphocyte) proliferation invivo or ex vivo. In one embodiment, the ex vivo expanded T lymphocyte isadministered to a subject in a method of adoptive immunotherapy.

In certain aspects, provided herein is a composition comprisingantigen-presenting cells that have been contacted in vitro with an AMHR2polypeptide, or a nucleic acid encoding an AMHR2 polypeptide, under acondition sufficient for an AMHR2 epitope to be presented by theantigen-presenting cells.

In some aspects, provided herein is a method for preparing lymphocytesspecific for AMHR2. The method comprises contacting lymphocytes with theantigen-presenting cells described above under conditions sufficient toproduce an AMHR2-specific lymphocyte capable of eliciting an immuneresponse against a cell that expresses AMHR2. Thus, theantigen-presenting cells also can be used to provide lymphocytes,including T lymphocytes and B lymphocytes, for eliciting an immuneresponse against cell that expresses AMHR2.

In some embodiments, a preparation of T lymphocytes is contacted withthe antigen-presenting cells described above for a period of time,(e.g., at least about 24 hours) to priming the T lymphocytes to an AMHR2epitope presented by the antigen-presenting cells.

In some embodiments, a population of antigen-presenting cells can beco-cultured with a heterogeneous population of peripheral blood Tlymphocytes together with an AMHR2 polypeptide, or a nucleic acidencoding an AMHR2 polypeptide. The cells can be co-cultured for a periodof time and under conditions sufficient for AMHR2 epitopes included inthe AMHR2 polypeptides to be presented by the antigen-presenting cellsand the antigen-presenting cells to prime a population of T lymphocytesto respond to cells that express a AMHR2. In certain embodiments,provided herein are T lymphocytes and B lymphocytes that are primed torespond to cells that express a AMHR2.

T lymphocytes can be obtained from any suitable source such asperipheral blood, spleen, and lymph nodes. The T lymphocytes can be usedas crude preparations or as partially purified or substantially purifiedpreparations, which can be obtained by standard techniques including,but not limited to, methods involving immunomagnetic or flow cytometrytechniques using antibodies.

In certain aspects, provided herein is a composition (e.g. apharmaceutical composition) comprising the antigen-presenting cells orthe lymphocytes described above, and a pharmaceutically acceptablecarrier and/or diluent. In some embodiments, the composition furthercomprises an adjuvant as described above.

In certain aspects, provided herein is a method for eliciting an immuneresponse to the cell that expresses AMHR2, the method comprisingadministering to the subject the antigen-presenting cells or thelymphocytes described above in effective amounts sufficient to elicitthe immune response. In some embodiments, provided herein is a methodfor treatment or prophylaxis of ovarian cancer, the method comprisingadministering to the subject an effective amount of theantigen-presenting cells or the lymphocytes described above. In oneembodiment, the antigen-presenting cells or the lymphocytes areadministered systemically, preferably by injection. Alternately, one canadminister locally rather than systemically, for example, via injectiondirectly into tissue (e.g., into tissue proximal to an ovarian cancertumor, such as into ovarian tissue), preferably in a depot or sustainedrelease formulation.

In certain embodiments, the antigen-primed antigen-presenting cellsdescribed herein and the antigen-specific T lymphocytes generated withthese antigen-presenting cells can be used as active compounds inimmunomodulating compositions for prophylactic or therapeutic treatmentof ovarian cancer. In some embodiments, the AMHR2-primedantigen-presenting cells described herein can be used for generatingCD8⁺ T lymphocytes, CD4⁺ T lymphocytes, and/or B lymphocytes foradoptive transfer to the subject. Thus, for example, AMHR2-specificlymphocyte can be adoptively transferred for therapeutic purposes insubjects afflicted with ovarian cancer.

In certain embodiments, the antigen-presenting cells and/or lymphocytesdescribed herein can be administered to a subject, either by themselvesor in combination, for eliciting an immune response, particularly foreliciting an immune response to cells that express a AMHR2. In someembodiments, the antigen-presenting cells and/or lymphocytes can bederived from the subject (i.e., autologous cells) or from a differentsubject that is MHC matched or mismatched with the subject (e.g.,allogeneic).

Single or multiple administrations of the antigen-presenting cells andlymphocytes can be carried out with cell numbers and treatment beingselected by the care provider (e.g., physician). In some embodiments,the antigen-presenting cells and/or lymphocytes are administered in apharmaceutically acceptable carrier. Suitable carriers can be the growthmedium in which the cells were grown, or any suitable buffering mediumsuch as phosphate buffered saline. The cells can be administered aloneor as an adjunct therapy in conjunction with other therapeutics.

EXEMPLIFICATION Experimental Procedures Generation of Recombinant MouseAMHR2 Polypeptides.

Total mRNA was extracted from ovaries of 8 week old female C57BL/6 mice.Primer pairs designed to amplify the AMHR2 sequence 1-140 or 170-568were used to generate cDNA encoding either the entire 140 amino acidextracellular domain or the entire 399 amino acid cytoplasmic domain ofmouse AMHR2 by RT-PCR. To optimize protein folding and enhance overallyield, substitutions for native codon sequences were made (Dapcel,Cleveland, Ohio), and the optimized cDNA was inserted into the NdeI-BamHI site of pET-3a (Novagen, Darmstadt, Germany) thereby providing aC-terminal 6×His-tagged (SEQ ID NO: 32) recombinant protein (FIGS. 1Aand 10A). E. coli were transformed with plasmids containing theseinserts. High level expression colonies were selected followinginduction with isopropylthiogalactopyronidase (IPTG; Amresco, SolonOhio) and were sequenced to confirm proper orientation and alignment.The 6×His-tagged (SEQ ID NO: 32) AMHR2 polypeptides were purified underdenaturing conditions using nickel-nitrilo triacetic acid (Ni-NTA)affinity chromatography (Qiagen Sciences, Germantown, Md.). The purifiedAMHR2 polypeptides were electrophoresed on denaturing SDS-PAGE gels(Bio-Rad, Hercules, Calif.) and blotted onto immunblot PVDF membrane(Bio-Rad). Immune detection of AMHR2 polypeptide was performed using theenhanced chemiluminescence system (Amersham Biosciences, Piscataway,N.J.) with HRP-conjugated His antibody (Qiagen). Prior to use, the6×His-tagged (SEQ ID NO: 32) AMHR2 polypeptides were purified by reversephase HPLC to yield endotoxin-free protein. Levels of endotoxin were<0.05 endotoxin units (<5 pg) per mg of recombinant protein.

Mice and Immunization.

Female C57BL/6 mice served as recipients of ID8 tumors. Mice wereobtained commercially (Jackson Laboratory, Bar Harbor, Me.) at six weeksof age and immunized at 7-10 weeks of age by subcutaneous injection inthe abdominal flanks with 100 μg of recombinant mouse AMHR2-CD orAMHR2-ED in 200 l of an emulsion of equal volumes of water and completeFreund's adjuvant (CFA; Difco, Detroit, Mich.) containing 400 μg ofMycobacteria tuberculosis. TgMISIIR-TAg (DR26 line) transgenic mice weremaintained by breeding male TgMISIIR-TAg (H-2b) mice to wild-typesyngeneic C57BL/6 females (Jackson Laboratory). TgMISIIR-TAg mice wereimmunized at 6-7 weeks of age with 100 μg of recombinant mouse AMHR2-CDor AMHR2-ED in CFA as described above. To determine fertilityphenotypes, age-matched test and control vaccinated C57BL/6 female micewere mated with the same C57BL/6 males.

Tumor Inoculation and Measurement.

ID8 cells were cultured in 75 or 225 cm² tissue culture flasks (BDBiosciences, Franklin Lakes, N.J.) in DMEM (Mediatech Cellgro, Manassas,Va.) containing 4% fetal bovine serum (Thermo Scientific Hyclone, Logan,Utah), 1% penicillin/streptomycin (Invitrogen, Carlsbad, Calif.), andinsulin-transferrin-sodium selenite media supplement (Sigma-Aldrich, St.Louis, Mo.) until the cells became 70-80% confluent. Cells wereharvested by trypsinization and washed twice with PBS. Female C57BL/6mice were inoculated subcutaneously in the left dorsal flank with 5×10⁶ID8 cells. Growth of ID8 tumors was assessed regularly by using aVernier caliper to measure length×width. Tumor growth endpoint wasdetermined by a measurement in any direction of 17 mm.

In Vivo Imaging and Measurement of Autochthonous Ovarian Tumors.

Bilateral ovarian tumor growth in female transgenic mice was measuredmonthly by ultrasound using the Vevo 770 high-resolution in vivomicro-imaging system for small animals (VisualSonics, Toronto, Canada).Real-time imaging of the abdomen was performed using the RMV704 lowfrequency probe/scan head and aqueous conductive gel after removing hairfrom the abdominal region. Anesthesia for immobilization wasadministered using a nose cone with continuous flow of 1-2% vol/volisoflurane during the image acquisition period lasting less than 30minutes, and oxygen supply was continuously maintained. The probe/scanhead was moved over the abdominal area very gently after applyingaqueous conductive gel. Measurements and calculation of tumor area wereperformed using the Vevo software B-Mode measurement tool allowing for a2-D assessment of ovarian tumor size in vivo with the polygon region ofinterest setting (VisualSonics). Measurement of solid tumor size byB-mode sonography has been shown to correlate well with histopathologicmeasurement.

RT-PCR.

Tissues were excised and stored frozen in RNA-Later (Life Technologies,Grand Island, N.Y.). RNA was either extracted from each tissue byhomogenization in TRIZOL reagent (Invitrogen), or purchased commercially(OriGene Technologies, Rockville, Md.; and ILS Biotech, Chestertown,Md.). cDNA was generated from bulk RNA using Superscript III(Invitrogen). Gene expression was quantified by qRT-PCR using SYBR GreenPCR mix (Applied Biosystems, Carlsbad, Calif.) with gene-specificprimers (Table 1). Relative gene expression was assessed bynormalization of each test gene expression level to β-actin expressionlevels in each individual tissue. Gene expression was determined byconventional RT-PCR using AMHR2- and β-actin-specific primers (Table 1).After amplification through 30 cycles, PCR products were separated onagarose gels (2% in 1 TBE buffer) and visualized under ultraviolet lightafter staining with ethidium bromide. Transgene expression in offspringof TgM1SIIR-TAg mice was determined by PCR amplification of a 773 bpfragment of SV40-TAg using primer pairs as previously described (Table1). DNA for the entire 140 amino acid sequence of the extracellulardomain of mouse AMHR2 was amplified by RT-PCR from mouse ovarian tissuesusing AMHR2-specific primer pairs (Table 1).

TABLE 1 Primer Pairs Used for Cloning, qRT-PCR, Detection of Transgene, and for Conventional  RT-PCR SEQ ID ProteinSequence (5′-3′) NO: AMHR2-ED F: AGCCCGAACCGCCGCCGCACCTGTG 31 (mouse)R: CCCCGGGGTAGCCTGCGGTTCCTGC  2 AMHR2-CD F: CTGAGCCGCTGTTCCGATTTGA  3(mouse) R: ATGTTGGGGCGCTTCCTCTCCT  4 AMHR2-CD F: CGGGCAGCTGCAAGGAAAAC  5(human) R: CCCCGGCTGGCAGTGATAAA  6 AMHR2-ED F: GCGGGGAAGCACAAAGACACT  7(mouse) R: CCGGCCATGGGTAAGATTCC  8 AMHR2-ED F: GGGGCTTTGGGCATTACTTCC  9(human) R: CCGGTCTTGGGTCAGGTTCC 10 AMHR2-CDF: GGATCCAAGGCCTGCAGAGTGCAAGGTG 11 R: AAGCTTCTACTCATTTACATACACCTG 12SV40-TAg F: TGCATGGTGTACAACATTCC 13 R: TTGGGACTGTGAATCAATGCC 14 IFNγF: GGATATCTGGAGGAACTGGCAA 15 R: TGATGGCCTGATTGTCTTTCAA 16 TNFαF: CGAGTGACAAGCCTGTAGCC 17 R: GTGGGTGAGGAGCACGTAGT 18 IL-1βF: AAGGAGAACCAAGCAACGACAAAA 19 R: TGGGGAACTCTGCAGACTCAAACT 20 IL-2F: GCAGGCCACAGAATTGAAAG 21 R: TCCACCACAGTTGCTGACTC 22 CD4F: ACACACCTGTGCAAGAAGCA 23 R: GCTCTTGTTGGTTGGGAATC 24 CD8F: TTACATCTGGGCACCCTTG 25 R: TTGCCTTCCTGTCTGACTAGC 26 β-ActinF: GGTCATCACTATTGGCAACG 27 R: ACGGATGTCAACGTCACACT 28 AMHR2F: GTATCCGCTGCCTCTACAGC 29 R: CAGAAGTCAGTGCCACAGGA 30

Flow Cytometry Analysis of Tumor Infiltrating Lymphocytes (TILs).

TILs were isolated from ID8 tumors by digestion of minced tumor for 30minutes at 37° C. in HBSS containing 50 KU of DNase I (Sigma-Aldrich)and 0.2 mg/ml collagenase II (Life Technologies) followed bydiscontinuous gradient centrifugation. The partially purified TILs weretreated with Fcγ III/II receptor antibody (BD Biosciences) in PBScontaining 0.5% BSA and 0.05% sodium azide and double-stained withFITC-conjugated anti-mouse CD3 and either PE-conjugated anti-mouse CD4or PE-conjugated anti-mouse CD8 (BD Biosciences). The CD3⁺ T cellpopulation was gated and analyzed for percentages of CD4⁺ and CD8⁺ Tcells. Data collected on 30,000 total events were analyzed using FlowJosoftware (BD Biosciences).

Passive Transfer of Tumor Immunity.

Ten days after immunization of female C57BL/6 mice with AMHR2polypeptide or control immunogen (ovalbumin; Sigma-Aldrich), LNCs at5×10⁶ cells/ml were activated in vitro with 20 ag/ml of immunogen in thepresence of IL-12 (10 ng/ml) and IL-18 (10 ng/ml; Peprotech, Rocky Hill,N.J.) in 24-well flat-bottom Falcon plates (BD Biosciences) in a totalvolume of 2.0 ml/well in DMEM supplemented as described above. After 3days of restimulation, 2×10⁷ activated whole LNCs were injectedintraperitoneally into sublethally γ-irradiated (5 Gy) naive femalerecipients. Alternatively, C57BL/6 female mice were immunized witheither AMHR2 polypeptide or OVA, and four weeks later, three groups ofcells were injected intraperitoneally into sublethally γ-irradiated (5Gy) naive female recipients including 7.5×10⁷ whole splenocytesreactivated with immunogen, IL-12, and IL-18 as described above, 2×10⁷similarly reactivated CD4⁺ T cells purified from whole splenocytes bymagnetic bead separation, and 2×10⁷ non-reactivated B220+ B cells alsopurified from whole splenocytes by magnetic bead separation. In allcases, hosts were inoculated subcutaneously on the day after celltransfer with 5×10⁶ ID8 cells, and tumor growth was assessed regularlyas described above. Purities of enriched cells were determined by flowcytometry analysis using CellQuest software (BD Biosciences) and wereconsistently found to be >90%.

Biostatistical Analysis.

Differences between mRNA expression levels and mean tumor weights werecompared using the Student's t-test. Differences between tumor growthcurves were compared by unweighted one-way ANOVA, and differences inmouse survival curves were compared using the log rank test.

Example 1: Generation of Recombinant Mouse AMHR2-CD

The longest hydrophilic domain of mouse AMHR2 consisting of the 170-568sequence comprising the 399 amino acids of the entire cytoplasmic domainwas expressed (FIG. 1A). The Ni-NTA affinity purified C-terminal6×His-tagged (SEQ ID NO: 32) protein migrated as a ˜44 kD protein asdetermined by Coomassie blue staining of an SDS-PAGE gel (FIG. 1B), andby Western blot immunostaining using HRP-conjugated His-specificantibody (FIG. 1C).

Example 2: Immunogenicity of AMHR2-CD

Ten days after AMHR2-CD immunization of female C57BL/6 mice, LNC showedproliferation in a dose response manner to AMHR2-CD but not torecombinant human cochlin, a control protein generated and purified in amanner similar to AMHR2-CD (FIG. 2A). This antigen-specificproliferation by LNC was elicited from purified CD4⁺ T cells but notfrom purified CD8⁺ T cells (FIG. 2B) and was inhibited by treatment ofcultures with CD4-specific but not CD8-specific antibodies (FIG. 2C).Four weeks after immunization, ELISA analysis of supernatants fromimmunogen-stimulated splenocytes showed a predominant proinflammatoryresponse to AMHR2-CD with high production of IFNγ and with relativelylow production of IL-2, IL-4, and IL-5 (FIG. 2D). Purification of T cellsubsets from the whole splenocyte population showed that CD4⁺ but notCD8⁺ T cells produced the IFNγ in response to AMHR2-CD (FIG. 2E). Twomonths after immunization, serum levels of AMHR2-CD-specific IgG weredetectable even at titers exceeding a 1:50,000 dilution (FIG. 2F).

Example 3: Benign Transient Ovarian Inflammation Following AMHR2-CDImmunization

The potential of AMHR2-CD immunization to induce ovarian autoimmunitywas next examined. Four and eight weeks after AMHR2-CD immunization ofC57BL/6 female mice, ovarian IFNγ gene expression was measured byqRT-PCR. Relative ovarian IFNγ gene expression was modestly elevated 4weeks after AMHR2-CD immunization but not after immunization with CFAalone (FIG. 3A). Eight weeks after immunization, relative ovarian IFNγgene expression was similar in both immunized groups of mice. Mostnotably, the transiently elevated IFNγ gene expression observed inAMHR2-CD immunized mice at 4 weeks were only 3 fold higher than CFAcontrol mice. No CD3⁺ T cell infiltrates were observed in ovaries byimmunohistochemical analysis at 4, 8, and 12 weeks after AMHR2-CDimmunization. The low transient expression of IFNγ in ovaries ofAMHR2-CD immunized mice was not associated with any detectable effect onovarian function as indicated by mouse fertility over four sequentialmating cycles during which no significant differences (P >0.60) occurredin the number of pups generated per litter between AMHR2-CD and CFAimmunized mice (FIG. 3C). AMHR2 gene expression was readily detected inthe ovaries and ID8 ovarian tumor cells and was not detected at anyappreciable levels in normal mouse uterus, stomach, spleen, heart, lung,kidney, and liver (FIG. 3C).

Example 4: Inhibition of Tumor Growth in Mice Immunized with AMHR2-CD

Whether vaccination with AMHR2-CD would inhibit growth of transplantableID8 tumors in C57BL/6 female mice was next determined. ID8 tumor growthwas inhibited in mice prophylactically vaccinated 15 days (FIG. 4A;P<0.001), 7 days (FIG. 4V; P<0.001), or 1 day (FIG. 4C; P<0.05) prior toinoculation of ID8 ovarian tumor cells. In addition, AMHR2-CDvaccination resulted in a significantly decreased overall tumor load asmeasured by final ID8 tumor weight at termination of experiments in micevaccinated 7 days (P<0.01) and 1 day (P<0.05) prior to ID8 inoculation(FIG. 4D). AMHR2-CD vaccination was also effective as therapy againstEOC. Vaccination with AMHR2-CD 60 days after inoculation of ID8 tumorssignificantly inhibited the growth of established, palpable ID8 tumors(P<0.05; FIG. 4E). Vaccination with AMHR2-CD significantly inhibited thegrowth of autochthonous EOCs that develop spontaneously in TgM1SIIR-TAgtransgenic mice (P<0.0001; FIG. 4F). Moreover, this inhibition in tumorgrowth was accompanied by a highly significant increased overallsurvival (OS) when compared to CFA vaccinated control mice (P<0.0005;FIG. 4G). This enhanced lifespan in AMHR2-CD vaccinated mice (mean191.25 days ±22.95) compared to CFA vaccinated control mice (mean 135days ±13.89) represents a 41.7% increase in OS.

At the termination of experiments, tumors were analyzed for inflammatoryinfiltrates. Immunohistochemical analysis consistently showed extensiveinfiltration of CD3+ T cells in tumors from AMHR2-CD vaccinated mice(FIG. 5A). No infiltrates were observed in tumors from mice immunizedwith CFA alone. Flow cytometry analysis of TILs showed a pronouncedincrease of CD4⁺ T cells in tumors from mice vaccinated with AMHR2-CDcompared to control mice immunized with CFA alone (40.7% vs. 11.7%; FIG.5b ). Substantial increases of CD8⁺ T cells in tumors did not occur inAMHR2-CD immunized mice compared to CFA immunized control mice (10.5%vs. 7.4%, respectively). Tumor RNA was next analyzed for gene expressionof proinflammatory factors by qRT-PCR. When compared to tumors from CFAimmunized control mice, tumors from AMHR2-CD immunized mice consistentlyshowed significantly increased relative gene expression (P<0.05 in allcases) for CD4, IFNγ, TNFα, and IL-2 but not for CD8 (FIG. 5C). Thesedata indicate the induction of a proinflammatory immune milieu withinthe ID8 tumor following immunization with AMHR2-CD.

Example 5: Passive Transfer of Tumor Immunity with CD4⁺ Cells

All recipient mice were inoculated with ID8 tumor cells on the day aftercell transfer. Tumor growth was significantly inhibited in micetransferred with AMHR2-CD-specific LNCs (P=0.04; FIG. 6A) andsplenocytes (P<0.01; FIG. 6B) when compared to mice receivingOVA-specific LNCs. At 190 days after transfer of primed splenocytes andtumor inoculation, mean tumor weights were significantly lower inrecipients of AMHR2-CD-specific splenocytes compared to recipients ofOVA-specific splenocytes (P<0.05; FIG. 6C). Transfer ofAMHR2-CD-specific CD4⁺ T cells purified from 4 week primed splenocytesresulted in significant inhibition of ID8 tumor growth compared totransfer of purified OVA-specific CD4⁺ T cells (P<0.0004; FIG. 6D)whereas transfer of AMHR2-CD-primed B220⁺ B cells purified from 4 weekprimed splenocytes did not significantly inhibit ID8 tumor growthcompared to transfer of OVA-primed B220⁺ B cells (P=0.07; FIG. 6D).Thus, AMHR2-CD-specific proinflammatory CD4⁺ T cells are sufficient fortransferring immune protection against the growth of EOC.

Example 6: Human AMHR2 Tissue Expression

The Human Protein Atlas database was examined to determine relativeAMHR2 gene expression in thirty human tissues. As seen in FIG. 7, AMHR2gene expression was observed to be above background in only five of thethirty tissues, with the highest expression observed in the ovary.

There are several known splice variants of AMHR2, with the varioussplice variants encoding different portions of AMHR2. The Human ProteinAtlas was next examined to determine the level of expression of thedifferent AMHR2 exons in the five tissues in which AMHR2 gene expressionwas observed. As seen in FIG. 8, expression of the exons encoding theextracellular domain of AMHR2 was confined exclusively to the ovary.Quantitative real-time RT-PCR (qRT-PCR) was used to show that expressionof AMHR2-ED is confined to the normal human ovary and is not expressedin the human adrenal gland or pancreas (FIG. 8B). In addition, as seenin FIG. 8D, human postmenopausal ovaries (mean 64 years; range 52-95years) and old mouse ovaries (nine months of age) have low levels ofAMHR2-ED expression when compared respectively to human premenopausalovaries (mean 31 years; range 27-45 years) and young mouse ovaries (6weeks of age). Moreover, all human EOC tumors that were examined showedhigh level expression of both AMHR2-ED transcripts (as assessed byqRT-PCR; FIG. 8E) and protein (as assessed by Western blot analysis;FIG. 8F). Thus, expression levels of both domains decline with age innormal ovaries, reducing the probability of even transient oophoritis invaccinating postmenopausal women with AMHR2 polypeptides.

Real-time RT-PCR was used to confirm that the AMHR2 extracellular domainwas expressed in normal human ovary, but not in the extracellular domain(FIG. 9A).

Whether the AMHR2 extracellular domain was expressed in EOCs was nexttested. When five EOCs were tested by western blot using an antibodyspecific for the AMHR2 extracellular domain, extracellular domainexpression was observed in all samples (FIG. 9B).

Example 7: Generation of Recombinant Mouse AMHR2-ED

The mouse AMHR2 extracellular domain made up of the N-terminal 140 aminoacids of the AMHR2 protein was expressed (FIG. 10A). The Ni-NTA affinitypurified C-terminal 6×His-tagged (SEQ ID NO: 32) protein migrated as a˜17 kD protein as determined by Coomassie blue staining of an SDS-PAGEgel and by Western blot immunostaining using HRP-conjugated His-specificantibody (FIG. 10B). One month after AMHR2-ED vaccination of young 6week old C57BL/6 female mice, ELISPOT analysis showed elevatedsplenocyte frequencies of IFNγ-producing antigen-specific T cells at amean of 1 per 3,831 splenocytes, and IL-17 at a mean of 1 per 21,739splenocytes, but not IL-5 (FIG. 10C).

The proinflammatory T cells responding to AMHR2-ED were predominantlyCD4+ T cells but also included CD8+ T cells producing IFNγ (FIG. 10D)and IL-17 (FIG. 10E). Four months after AMHR2-ED vaccination, serum IgGantibody responses against AMHR2-ED were detectable at dilutions up to1/50,000 (FIG. 10F) and consisted predominantly of IgG1 and IgG2bisotypes (FIG. 10G). AMHR2-ED was highly immunogenic in several mousestrains showing completely divergent major histocompatibility complexhaplotypes including C57BL/6 (H-2b), BALB/c (H-2^(d)), A/J (H-2^(a)),and FVBN/J (H-2^(q)) in which IFNγ secreting splenocytes reachedfrequencies one month after vaccination with means of 1 per 4,310, 1 per4,425, 1 per 8,772, and 1 per 10,000, respectively (FIG. 10H).

Example 8: Immunogenicity of AMHR2-ED

The immunogenicity of AMHR2-ED was determined by immunization ofTgM1SIIR-TAg (DR26 line) transgenic female mice with AMHR2-ED in CFA.Four weeks after immunization, splenocytes from AMHR2-ED showedantigen-specific recall proliferative responses to AMHR2-ED but not torecombinant mouse β-casein, an irrelevant control antigen generated andpurified in a manner similar to AMHR2-ED (FIG. 11A). In contrast,splenocytes from CFA-immunized mice were unresponsive to both AMHR2-EDand β-casein (FIG. 11B). ELISA analysis of culture supernatants showedAMHR2-ED activated production of high levels of the proinflammatorycytokines IFNγ and IL-17, and minimal production of the type-2regulatory cytokine, IL-5 (FIG. 11C). Splenocytes from AMHR2-EDimmunized mice showed significantly high frequencies of type-1 (˜1/4,000lymphocytes; p<0.0001) and type-17 (˜1/20,000 lymphocytes; p<0.0²)proinflammatory T cells but minimal frequencies of type-2 regulatory Tcells expressing IL-5 (FIG. 11D). Four months after immunization withAMHR2-ED, serum titers for AMHR2-ED specific IgG were still detectableat titers exceeding 1/50,000 dilutions (p<0.001, FIG. 11E). Theseresults indicate that AMHR2-ED is highly immunogenic.

To further evaluate the nature of the immune response generated byvaccination with AMHR2-ED, TgM1SIIR-TAg transgenic female mice wereimmunized with either AMHR2-ED at 25 jag/ml or 50 jag/ml. One monthafter immunization, spleens were harvested and IFNγ and IL-17 expressiondetected. As seen in FIG. 12A-B, splenocytes and purified CD4⁺ but notCD8+ T cells isolated one month after immunization of with AMHR2-EDshowed a prominent antigen-specific induction of type-1 and type-17proinflammatory T cells. Immunohistochemical analysis of autochthonousEOC taken from 7 month old female TgMISIIR-Tag mice that were immunizedat 6 weeks of age with AMHR2-ED showed predominant infiltration of CD3+T cells and CD4+ T cells, but not CD8+ T cells (FIG. 12C-D).

Example 9: Immunization with AMHR2-ED does not Affect Fertility

The level of ovarian inflammation following immunization with AMHR2-EDwas examined. Ovaries of mice were harvested four weeks and 8 weeksfollowing immunization with AMHR2-ED and analyzed by RT-PCR. As seen inFIG. 13, harvested ovaries showed expression of the inflammatorycytokine IL-10 at four weeks but not at eight weeks followingimmunization. IFNγ expression was not elevated at either time pointfollowing immunization. In contrast, elevated gene expression did notoccur for IL-1β or IFNγ in ovaries from 9 month old C57BL/6 female miceat 4 weeks after AMHR2-ED vaccination (FIG. 13C).

The fertility of mice immunized with AMHR2-ED was next examined. As seenin FIG. 14, ovarian function as measured by fertility over four matingcycles was unaffected by AMHR2-ED immunization. Specifically, nosignificant differences in mean number of pups per litter or mean pupbirth weights were detected between mice immunized with AMHR2-ED in CFAor control mice immunized with CFA alone.

Example 10: Immunization with AMHR2-ED Inhibits Growth of Ovarian CancerTumors

The ability of AMHR2-ED vaccination to inhibit growth of authochthonousand transplantable ovarian tumors was tested. As seen in FIG. 15A-B,prophylactic AMHR2-EC vaccination of female TgM1SIIR-TAg transgenic miceat 6-7 weeks of age resulted in a highly significant inhibition ingrowth of autochthonous EOC (p<0.0001) and a 42% increased overallsurvival compared to control mice vaccinated with CFA alone (mean193.7±34.5 days vs. mean 135±13.89 days). Similar significant inhibitionin growth of transplantable TgMISIIR EOC tumors occurred in micevaccinated either 7 days or15 days prior to inoculation with 3×10⁶ mouseovarian carcinoma (MOVCAR) cells (p<0.001; FIG. 15C-D).

Similarly, prophylactic AMHR2-ED vaccination of 6-7 week oldTgMISIIR-TAg (DR26) transgenic mice significantly delayed the appearanceand growth of autochthonous EOC compared to control mice vaccinated withCFA alone (P<0.001; FIG. 15E). This inhibition in the emergence andgrowth of autochthonous EOC resulted in a highly significant 42%increased overall survival (P<0.0001) compared to control micevaccinated with CFA alone (mean 194±35 days vs. mean 135±14 days,respectively; FIG. 15F). AMHR2-ED vaccination was also effective inproviding highly significant immunotherapy against EOC in TgMISIIR-TAg(DR26) transgenic mice with established growing autochthonous EOC(P<0.0001; FIG. 15G). Immunohistochemical analysis of autochthonous EOCfrom TgMISIIR-TAg (DR26) mice vaccinated with AMHR2 consistently showedprominent infiltrates of CD3+ T cells (FIG. 15H, upper left panel) andCD4+ T cells (FIG. 15H, upper middle panel) with occasional CD8+ T cells(FIG. 15H, upper right panel). Corresponding immunostained EOC fromcontrol mice vaccinated with CFA alone consistently failed to showdetectable T cell infiltrates (FIG. 3f , lower panels).

Example 11: Transfer of AMHR2-ED Primed T Cells and B Cells InhibitsGrowth of Ovarian Cancer Tumors

The effect of transfer of AMHR2-ED primed CD4⁺ T cells and B220⁺ B cellson EOC tumor growth was tested. As seen in FIG. 16, inhibition of growthof MOVCAR EOCs occurred following transfer of CD4⁺ T cells or B220⁺ Bcells from mice immunized with AMHR2-ED. What is more, as seen in FIG.17, the transfer of CD4⁺ T cells or B220⁺ B cells from AMHR2-EDimmunized mice also increased overall survival in MOVCAR tumor bearingmice that received cells. Transfer of cells into naïve recipients andinoculation of MOVCAR EOCs occurred within one day of each other.Additionally, as seen in FIG. 18, growth inhibition of ID8-VEGF EOCtumors occurred in mice receiving CD4+ T cells (upper left panel) orsera (lower left panel) from mice immunized with AMHR2-ED. Enhancedoverall survival occurred in mice receiving CD4+ T cells (upper rightpanel) or sera (lower right panel) from mice immunized with AMHR2-ED.Transfer of cells into naïve recipients and inoculation of ID8-VEGF EOCsoccurred within one day of each other. Asterisks indicate significance.

Example 12: Passive Transfer of Tumor Immunity with CD4+ T Cells, B220+B Cells, and IgG

The identity of the immune population that accounted for inhibition ofEOC tumor growth was tested. It was found that transfer of AMHR2-EDprimed CD4+ T cells into TgMISIIR-TAg (low) female mice one day prior toMOVCAR inoculation resulted in highly significant inhibition of tumorgrowth (P<0.0001; FIG. 20A) and enhanced overall survival (P<0.006; FIG.20B) compared to mice receiving ovalbumin primed CD4+ T cells. Moreover,transfer of AMHR2-ED primed B220+ B cells into TgMISIIR-TAg (low) femalemice one day prior to MOVCAR inoculation also mediated significantinhibition of tumor growth (P<0.0001; FIG. 20C) and enhanced overallsurvival (P<0.009; FIG. 20D) compared to mice receiving B220+ B cellsfrom ovalbumin immunized mice. In addition, transfer of affinitypurified IgG from AMHR2-ED immunized mice into TgMISIIR-TAg (low) femalemice one day prior to MOVCAR inoculation resulted in significantinhibition of tumor growth (P<0.0001; FIG. 20E) and enhanced overallsurvival (P<0.002; FIG. 20F) compared to mice receiving affinitypurified IgG from ovalbumin immunized mice.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments described herein. Such equivalents are intended to beencompassed by the following claims.

We claim:
 1. A method of treating an ovarian cancer tumor in a femalehuman subject comprising administering to the subject an immunogeniccomposition comprising an Anti-Mullerian Hormone Receptor, Type II(AMHR2) polypeptide, wherein the AMHR2 polypeptide has an amino acidsequence that comprises at least 8 consecutive amino acids of SEQ IDNO:
 1. 2. The method of claim 1, wherein the at least 8 consecutiveamino acids are identical to an amino acid sequence in the cytoplasmicdomain of AMHR2.
 3. The method of claim 2, wherein the polypeptide hasan amino acid sequence that comprises 100 consecutive amino acids thatare at least 80% identical to an amino acid sequence in the cytoplasmicdomain of AMHR2.
 4. The method of claim 3, wherein the polypeptide hasan amino acid sequence that comprises 100 consecutive amino acidsidentical to an amino acid sequence in the cytoplasmic domain of AMHR2.5. The method of claim 4, wherein the polypeptide has an amino acidsequence identical to the amino acid sequence of the cytoplasmic domainof AMHR2.
 6. The method of claim 2, wherein the polypeptide does notcomprise an amino acid sequence identical to the extracellular domain ofAMHR2.
 7. The method of claim 2, wherein the polypeptide does notcomprise an amino acid sequence identical to the transmembrane domain ofAMHR2.
 8. The method of claim 1, wherein the at least 8 consecutiveamino acids are identical to an amino acid sequence in the extracellulardomain of AMHR2.
 9. The method of claim 8, wherein the polypeptide hasan amino acid sequence that comprises 100 consecutive amino acids thatare at least 80% identical to an amino acid sequence in theextracellular domain of AMHR2.
 10. The method of claim 9, wherein thepolypeptide has an amino acid sequence that comprises 100 consecutiveamino acids identical to an amino acid sequence in the extracellulardomain of AMHR2.
 11. The method of claim 10, wherein the polypeptide hasan amino acid sequence identical to the amino acid sequence of theextracellular domain of AMHR2.
 12. The method of claim 8, wherein thepolypeptide does not comprise an amino acid sequence identical to thetransmembrane domain of AMHR2.
 13. The method of claim 8, wherein thepolypeptide does not comprise an amino acid sequence identical to thecytoplasmic domain of AMHR2.
 14. The method of claim 1, wherein theimmunogenic composition comprises an adjuvant.
 15. The method of claim14, wherein the adjuvant is selected from the group consisting ofAdjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calciumphosphate, β-Glucan Peptide, CpG DNA, GM-CSF, GPI-0100, IFA, IFN-γ,IL-17, lipid A, lipopolysaccharide, Lipovant, Montanide,N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, poly-IC, quil A,trehalose dimycolate and zymosan.
 16. The method of claim 14, whereinthe adjuvant is one that induces a mixed type 1/type 17 immune response.17-18. (canceled)
 19. The method of claim 1, wherein the ovarian cancertumor is epithelial ovarian cancer (EOC) tumor.
 20. The method of claim1, wherein the ovarian cancer tumor expresses AMHR2.
 21. (canceled) 22.The method of claim 1, further comprising the step of determiningwhether the ovarian cancer tumor expresses AMHR2. 23-63. (canceled) 64.A kit for preventing or treating ovarian cancer in a female humansubject comprising an Anti-Mullerian Hormone Receptor, Type II (AMHR2)polypeptide, wherein the AMHR2 polypeptide has an amino acid sequencethat comprises at least 8 consecutive amino acids of SEQ ID NO: 1.65-83. (canceled)